Modulatory proteins of human CNS receptors

ABSTRACT

Neurotransmission by excitatory amino acids (EAAs) such as glutamate is mediated via membrane-bound surface receptors. This neurotransmission has been found to be modulated by certain modulatory proteins. DNA coding for a family of such modulatory proteins has now been isolated and the modulatory proteins have been characterized. Herein described are recombinant cell lines which produce these modulatory proteins as heterologous membrane-bound products. Also described are related aspects of the invention, which are of commercial significance, including the use of cell lines which express the modulatory proteins either homomerically, or heteromerically in a complex with an NMDA receptor, as a tool for discovery of compounds which affect the function of the modulatory proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. 07/987,953, filed Dec. 11, 1992.

FIELD OF THE INVENTION

[0002] This invention relates to the application of recombinant DNAtechnology in the field of neurobiology. More particularly, theinvention relates to the cloning and expression of DNA coding forproteins which modulate the function of glutamate receptors.

BACKGROUND OF THE INVENTION

[0003] In the mammalian central nervous system (CNS), the transmissionof nerve impulses is controlled by the interaction between aneurotransmitter substance released by the “sending” neuron which thenbinds to a surface receptor on the “receiving” neuron, to causeexcitation thereof. L-glutamate is the most abundant neurotransmitter inthe CNS, and mediates the major excitatory pathway in vertebrates.Glutamate is therefore referred to as an excitatory amino acid (EAA) andthe receptors which respond to it are variously referred to as glutamatereceptors, or more commonly as EAA receptors.

[0004] Members of the EAA receptor family can be grouped into three maintypes based on differential binding to certain glutamate analogs. Onetype of EAA receptor, which in addition to glutamate also binds thecompound NMDA (N-methyl-D-aspartate), is referred to as the NMDA type ofEAA receptor. Two other glutamate-binding types of EAA receptor, whichdo not bind NMDA, are named according to their preference for bindingwith two other EAA receptor agonists, namely AMPA(alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate), and kainate(2-carboxy-4-(1-methylethenyl)-3-pyrrolidineacetate). Accordingly,receptors which bind glutamate but not NMDA, and which bind with greateraffinity to kainate than to AMPA, are referred to as kainate-type EAAreceptors. Similarly, those EAA receptors which bind glutamate but notNMDA, and which bind AMPA with greater affinity than kainate arereferred to as AMPA-type EAA receptors.

[0005] The glutamate-binding EAA receptor family is of greatphysiological and medical importance. Glutamateis involved in manyaspects of long-term potentiation (learning and memory), in thedevelopment of synaptic plasticity, in epileptic seizures, in neuronaldamage caused by ischemia following stroke or other hypoxic events, aswell as in other forms of neurodegenerative processes. The developmentof therapeutics which modulate these processes is being slowed by thelack of any homogeneous source of receptor material with which todiscover selectively binding drug molecules, which interact specificallyat the interface of an appropriate EAA receptor. The brain derivedtissues currently used to screen candidate drugs are heterogeneousreceptor sources, possessing on their surface many receptor types whichinterfere with studies of the EAA receptor/ligand interface of interest.The search for human therapeutics is further complicated by the limitedavailability of brain tissue of human origin. It would therefore bedesirable to obtain cells that are genetically engineered to produceonly the receptor of interest. With cell lines expressing clonedreceptor cDNA, a substrate which is homogeneous for the desired receptoris provided, for drug screening programs.

[0006] Non-human cDNAs which appear to encode the NMDA-type of EAAreceptor have recently been identified and isolated. A cDNA encoding asubunit polypeptide of an NMDA receptor in rat, designated NR1, has beenisolated as described by Moriyoshi et al. in Nature 354: 31, 1991. Anextension of this work has revealed seven isoforms of NR1, presumablygenerated by combinations of alternative RNA splicing in the amino- andcarboxy-terminal regions of NR1 (Anantharam et al. FEBS Lett. 305: 27,1992; Durand et al. Proc. Natl. Acad. Sci. USA 89: 9359, 1992; Nakanishiet al. Proc. Natl. Acad. Sci. USA 89: 8552, 1992; Sugihara et al.Biochem. Biophys. Res. Commun. 185: 826, 1992; Hollmann et al. Neuron10: 943, 1993; Kusiak and Norton. Mol. Brain. Res. 20: 64, 1993). DNAencoding NR1 and one of its isoforms have also been cloned from mousebrain by Yamazaki et al. as described in FEBS Lett. 300: 39, 1992. Otherrat NMDA receptor subunits, designated NR2A, NR2B, NR2C and NR2D, havealso been identified (Monyer et al. Science 256: 1217, 1992; Ishii etal. J. Biol. Chem. 268: 2836, 1993), as well as mouse NMDA receptorsubunits which have been designated ε1, ε2, ε3 and ε4 (Meguro et al.Nature 357: 70, 1992; Kutsuwada et al. Nature 358: 36, 1992; Ikeda etal. FEBS Lett. 313: 34, 1992).

[0007] There has emerged from these molecular cloning advances, a betterunderstanding of the structural features of NMDA receptors and theirsubunits, as they exist in the non-human brain. According to the currentmodel, each NMDA receptor is heteromeric, consisting of individualmembrane-anchored subunits, each comprising transmembrane regions andextracellular domains that dictate ligand-binding properties andcontribute to the ion-gating function served by the receptor complex.

[0008] In the search for therapeutics useful to treat CNS disorders inhumans, it is highly desirable to obtain knowledge of human EAAreceptors, and proteins which modulate the activity of these receptors.Such an understanding would provide a means to screen for compounds thatselectively interact with this activity, i.e. to stimulate or inhibitreceptor activity, thereby providing a means to identify compoundshaving potential therapeutic utility in humans. Non-human mammalianmodels are not suitable for this purpose despite significant proteinhomology due to the fact that minute sequence discrepancies have beenfound to cause dramatic pharmacological and functional variation betweenspecies homologues of the same protein (Oksenberg et al., Nature,360:161, 1992; Hall et al. Trends Pharmacol. Sci. 14: 376, 1993). It istherefore particularly desirable to provide cloned cDNA encoding humanEAA receptor proteins or modulatory proteins thereof, and cell linesexpressing these proteins, in order to generate a screening method forcompounds therapeutically useful in humans. These, accordingly, areobjects of the present invention.

SUMMARY OF THE INVENTION

[0009] Human cDNAs encoding NMDA receptor modulatory proteins have beenidentified and characterized, and include proteins referred to herein asthe NR3 and NR4 modulatory proteins. Specifically encompassed are parentproteins designated the NR3-1 and NR4-1 proteins, as well as functionalsequence-related variants of NR3-1 and NR4-1, and functional fragmentsof NR3-1 and NR4-1.

[0010] In one of its aspects, thus, the present invention provides anisolated polynucleotide, consisting either of DNA or of RNA, which codesfor a human NR3 protein, or functional fragments thereof.

[0011] In another aspect of the present invention, there is provided acell that has been genetically engineered to produce a human EAAreceptor modulatory protein belonging to the herein-defined NR3 family.In related aspects of the present invention, there are providedrecombinant DNA constructs and methods useful to obtain substantiallyhomogeneous sources of the human NR3 protein, comprising the steps ofculturing the genetically engineered cells, and then recovering thecultured cells.

[0012] In another aspect of the present invention, there is provided amethod for evaluating interaction between a candidate ligand and a humanEAA receptor modulatory protein, which comprises the steps of incubatingthe candidate ligand with a genetically engineered cell as describedabove, or with a membrane preparation derived therefrom, and thenassessing said interaction by determining the extent of protein/ligandbinding, or by determining the ligand-induced electrical current acrosssaid cell.

[0013] In yet another aspect of the present invention, a cell that hasbeen engineered genetically to produce a human heteromeric NR3/receptorcomplex comprising an NR3 protein and an NMDA receptor is provided.

[0014] In a further aspect of the present invention, there is provided amethod for evaluating interaction between a candidate ligand and a humanheteromeric NR3/receptor complex comprising an NR3 protein and an NMDAreceptor, said method comprising the steps of incubating the candidateligand with a cell line engineered to produce said receptor complex, orwith a membrane preparation derived therefrom, and then assessing theinteraction therebetween by determining the extent of protein/ligandbinding, or by determining the ligand-induced electrical current acrosssaid cell.

[0015] Other aspects of the present invention include a human NR3protein, in a form essentially free from other proteins of human origin,functional and immunogenic fragments of the protein, antibodies whichbind to the protein, and oligonucleotides which hybridize to nucleicacid encoding the protein.

[0016] Other aspects of the present invention, which encompass variousapplications of the discoveries herein described, will become apparentfrom the following detailed description, and from the accompanyingdrawings in which:

BRIEF REFERENCE TO THE DRAWINGS

[0017]FIG. 1 provides the nucleotide sequence (SEQ ID NO: 1) of DNAencoding an NR3 modulatory protein, and the deduced amino acid sequence(SEQ ID NO: 2) thereof;

[0018]FIGS. 2A and 2B illustrate, with plasmid maps, the strategy usedto construct expression vectors harbouring the DNA sequence illustratedin FIG. 1;

[0019]FIG. 3 provides, with reference to FIG. 1, the partial DNA andamino acid sequences (SEQ ID NOs: 5 & 6) of a naturally occurringvariant of the modulatory protein illustrated in FIG. 1;

[0020]FIG. 4 provides the partial nucleotide sequence (SEQ ID NO: 7) ofDNA encoding an NR4 modulatory protein, and the deduced amino acidsequence (SEQ ID NO: 8) thereof;

[0021]FIG. 5 provides the nucleotide sequence (SEQ ID NO: 9) of DNAencoding the NR2A-1 probe;

[0022]FIG. 6 provides the nucleotide sequence (SEQ ID NO: 10) of DNAencoding the NMDAR1-1 receptor, and the deduced amino acid sequence (SEQID NO: 11) thereof;

[0023]FIG. 7 provides a comparison of partial nucleotide sequences ofNMDAR1-1 (SEQ ID NO: 12) with its variants, NMDAR1-2, NMDAR1-3A andNMDAR1-3C (SEQ ID NOs: 13, 14 & 15, respectively);

[0024]FIG. 8 provides a comparison of the amino acid sequences ofNMDAR1-1 (SEQ ID NO: 16) and NMDAR1-4 (SEQ ID NO: 17);

[0025]FIG. 9 provides a comparison of the amino acid sequences ofNMDAR1-1/2/3/4 (SEQ ID NO: 18) and NMDAR1-5/6/7/8 (SEQ ID NO: 19); and

[0026] FIGS. 10A-10D graphically illustrate electrophysiologicalproperties of a heteromeric complex comprising NR3-1 and NMDAR1-3C.

DETAILED DESCRIPTION OF THE INVENTION AND ITS PREFERRED EMBODIMENTS

[0027] The present invention relates to modulatory proteins ofexcitatory amino acid (EAA) receptors of human origin, and to isolatedpolynucleotides encoding them. More particularly, the present inventionis directed to novel human modulatory proteins, herein designated theNR3 and NR4 EAA receptor modulatory proteins, which modulate theactivity of human EAA receptors of the NMDA-type. The NR3 and NR4modulatory proteins comprise the NR3-1 and NR4-1 parent proteins, aswell as functional sequence-related variants of the human NR3-1 proteinand functional fragments of the NR3-1 protein.

[0028] As used herein, the term “modulatory protein” refers to a proteinthat, when combined with a human EAA receptor, and in particular with ahuman NMDA receptor, forms a heteromeric receptor complex havingelectrophysiological properties which are distinct from theelectrophysiological properties of a homomeric receptor complex formedfrom the selected NMDA receptor alone. Thus, the NR3 and NR4 proteins ofthe present invention have been found to modulate the ion channelactivity of NMDA-type receptors, i.e. receptors having a ligand bindingprofile comprising specific binding affinity for glutamate, NMDA andMK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-iminemaleate]. The electrophysiological properties, or ion channel activity,of EAA receptors is typically determined using establishedelectrophysiological assays appropriate for detecting conductance acrossa cell membrane such as the assay described by Hollmann et al. in Nature342: 643.

[0029] The term “isolated” as it is used herein with respect toNR3-encoding polynucleotides refers to polynucleotides which are freefrom human DNA which encodes, or partially encodes, CNS proteins otherthan NR3 proteins and NMDA receptor proteins.

[0030] The term “heteromeric receptor complex” is used to refer to areceptor complex comprising a modulatory protein, in accordance with thepresent invention, and an NMDA receptor. A “heteromeric NR3/receptorcomplex” refers to a receptor complex comprising an NR3 modulatoryprotein and an NMDA receptor.

[0031] Variants of the NR3-1 and NR4-1 parent modulatory proteins alsoform modulatory proteins as defined above. Specifically included arefunctional variants which exhibit a modulatory activity similar to thatof the parent protein, and which demonstrate substantial sequencehomology to the parent protein. Specifically, variants of NR3-1 willshare greater than 98.5% amino acid identity with the NR3-1 protein.Variants of the NR3-1 protein include both naturally occurring variants,an example of which is the NR3-2 protein, illustrated in part in FIG. 3by nucleic acid and amino acid sequence (SEQ ID NOs: 5 & 6), as well assynthetically derived variants of the human NR3-1 protein.

[0032] The term “fragment” is used herein to denote functional segmentsof an NR3 or NR4 protein.

[0033] Variants and fragments of the NR3 and NR4 proteins are said to be“functional” if, on coexpression with an NMDA receptor in a heteromericreceptor complex as defined above, the complex, when assayedelectrophysiologically, exhibits ligand-induced ion channel activityhaving measurable current, i.e. current which is greater than thecurrent in the absence of the ligand or greater than the “baseline”current, and the channel activity possess properties which arecharacteristic of an NMDA ion channel, for example the channel activityis blocked by Mg⁺⁺ ions and by MK-801.

[0034] Each of the naturally occurring members of the human NR3 and NR4modulatory proteins possess structural features similar to those of EAAreceptors, including an extracellular amino-terminal (N-terminal)region, as well as internal hydrophobic domains which serve to anchorthe protein within the cell surface membrane. The particular human EAAreceptor modulatory protein designated NR3-1 is a protein characterizedstructurally as a single polypeptide chain that is produced initially inprecursor form bearing an N-terminal signal peptide, and is transportedto the cell surface in mature form, lacking the signal peptide. TheNR3-1 protein, including its signal peptide, consists of 1,484 aminoacids arranged in the sequence illustrated, by single letter code, inFIG. 1 (SEQ ID NO: 2). The particular human EAA receptor modulatoryprotein designated NR4-1 is a protein encoded by the partial nucleotidesequence illustrated in FIG. 4 (SEQ ID NO: 7).

[0035] A naturally occurring structurally-related variant of the NR3-1protein has also been identified and is designated herein, the NR3-2modulatory protein. This variant protein differs from its NR3-1 parentby a single amino acid as illustrated in FIG. 3. Specifically, theserine residue at position 407 in NR3-1 is an asparagine residue in theNR3-2 variant. This change is reflected as a single nucleotidedifference between the nucleic acid encoding the two proteins, namely acodon change from “AGC” in NR3-1 to “AAC” in NR3-2.

[0036] The NR3 and NR4 proteins are characterized by their modulatoryactivity particularly with respect to human NMDA-type receptors, andmore particularly with respect to NMDA receptors of the NMDAR1 family,which are described in detail in co-pending U.S. patent appln. Ser. No.07/987,953, the content of which is incorporated herein by reference.The NMDAR1 family of EAA receptors comprises the NMDAR1-1 receptor, thenucleic acid sequence of which is illustrated in FIG. 6 (SEQ ID NO: 10),and variants of the NMDAR1-1 receptor which retain an NMDA-type ligandbinding profile and which are structurally related to NMDAR1-1, i.e.share at least 99.6% amino acid identity with the 1-845 amino acidregion of the NMDAR1-1 receptor, and preferably share 100% amino acididentity in this region. There are both naturally occurring andsynthetically derived variants of the human NMDAR1-1 receptor. Naturallyoccurring variants include, but are not restricted to, receptor variantsdesignated human NMDAR1-2, NMDAR1-3A and NMDAR1-3C, the partialnucleotide sequences of which are illustrated in FIG. 7 (SEQ ID NOs: 13,14 & 15, respectively) and compared to the nucleotide sequence ofNMDAR1-1 (SEQ ID NO: 12). Another variant, designated NMDAR1-3B, differsin amino acid sequence from the NMDAR1-1 and NMDAR1-3C receptors by asingle amino acid at position 470. This amino acid is lysine inNMDAR1-3B and is glutamic acid in NMDAR1-1 and NMDAR1-3C. This changeresults from a single base pair change in the codon at position 2560 ofNMDAR1-1 and NMDAR1-3C from GAG to AAG in the 3B variant. An NMDAR1-4variant differs from the NMDAR1-1 receptor by a peptide insert betweenamino acids 845 and 846 of NMDAR1-1 as illustrated in FIG. 8. Furthervariants include NMDAR1-4, NMDAR1-5, NMDAR1-6, NMDAR1-7 and NMDAR1-8,which correspond respectively to the NMDAR1-1, NMDAR1-2, NMDAR1-3 andNMDAR1-4 receptors additionally including a 21 amino acid insert asillustrated in FIG. 9.

[0037] One of skill in the art will appreciate that variants of any oneof the NMDAR1-1 to NMDAR1-8 receptors which include minor variationsfrom the amino acid sequences thereof, e.g. 1 to 6 amino acidsubstitutions, deletions or additions, and resulting in receptorsretaining the ligand binding profile characteristic of NMDA-typereceptors, are also encompassed within the NMDAR1 family of receptors.

[0038] Accordingly, the NR3 and NR4 proteins of the present inventionare useful in a heteromeric structure to screen for candidate compoundshaving the ability to alter the activity of the heteromeric receptorcomplex. In addition, and despite the understanding that the NR3 and NR4proteins require a heteromeric structure to function in a modulatorysense, cells producing NR3 and NR4 proteins homomerically, independentof association with an NMDA receptor, can be exploited for the purposeof screening candidate ligands for the ability to interact specificallytherewith. Those compounds found to interact with an NR3 or an NR4protein represent potential drug compounds which may have agonist orantagonist properties useful in the treatment of neurological diseaseconditions.

[0039] For use in assessing interaction between an NR3 or an NR4protein, either in homomeric or heteromeric form, and a candidatecompound, it is desirable to construct by application of geneticengineering techniques a cell that produces a human NR3 or NR4 proteinin functional form as a heterologous product. The construction of suchcell lines is achieved by introducing into a selected host cell arecombinant DNA construct in which DNA coding for a secretable form ofthe modulatory protein, i.e. a form bearing either its native signalpeptide or a functional, heterologous equivalent thereof, is associatedwith expression controlling elements that are functional in the selectedhost to drive expression of the modulatory protein-encoding DNA, andthus elaborate the desired NR3 or NR4 protein. Such cells are hereincharacterized as having the protein-encoding DNA incorporated“expressibly” therein. The protein-encoding DNA is referred to as“heterologous” with respect to the particular cellular host if such DNAis not naturally found in the particular host.

[0040] It is most desirable to use a mammalian cell host to produce thepresent modulatory proteins due to their human origin; however, othersuitably engineered eukaryotic and prokaryotic hosts may also beemployed to produce NR3 proteins. Accordingly, bacterial hosts such asE. coli and B. subtilis, fungal hosts such as Asgergillus and yeast andinsect cell hosts such as Spodoptera frugiperda, are examples ofnon-mammalian hosts that may also be used to produce NR3/NR4 proteins ofthe present invention.

[0041] The particular cell type selected to serve as host for productionof the human modulatory proteins can be any of several cell typescurrently available in the art. Preferably, where the modulatory proteinwill be expressed in heteromeric form, i.e. in conjunction with an NMDAreceptor, the cell type selected will not be one which in its naturalstate elaborates a surface receptor that has ion channel activity orthat elaborates a protein that is capable of modulating receptoractivity, so as to confuse the assay results sought from the engineeredcell line. Generally, such problems are avoided by selecting as host anon-neuronal cell type. However, neuronal cells may nevertheless serveas expression hosts, provided that any “background” activity isaccounted for in the assay results.

[0042] According to one embodiment of the present invention, the cellline selected to serve as host for modulatory protein production is amammalian cell. Several types of such cell lines are currently availablefor genetic engineering work, and these include the chinese hamsterovary (CHO) cells for example of K1 lineage (ATCC CCL 61) including thePro5 variant (ATCC CRL 1281); fibroblast-like cells derived fromSV40-transformed African Green monkey kidney of the CV-1 lineage (ATCCCCL 70), of the COS-1 lineage (ATCC CRL 1650) and of the COS-7 lineage(ATCC CRL 1651); murine L-cells, murine 3T3 cells (ATCC CRL 1658),murine C127 cells, human embryonic kidney cells of the 293 lineage (ATCCCRL 1573), human carcinoma cells including those of the HeLa lineage(ATCC CCL 2), and neuroblastoma cells of the lines IMR-32 (ATCC CCL127), SK-N-MC (ATCC HTB 10) and SK-N-SH (ATCC HTB 11).

[0043] A variety of gene expression systems have been adapted for usewith these hosts and are now commercially available. Any one of thesesystems can be exploited to drive expression of DNA encoding NR3 or NR4proteins. These systems, available typically in the form of plasmidicvectors, incorporate expression cassettes, the functional components ofwhich include DNA constituting host-recognizable expression controllingsequences which enable expression of the receptor-encoding DNA whenlinked 5′ thereof. The systems further incorporate DNA sequences whichterminate expression when linked 3′ of the protein-encoding region.Thus, for expression in a selected mammalian cell host, there isgenerated a recombinant DNA expression construct in which DNA encodingan NR3 or NR4 protein is linked with expression controlling DNAsequences recognized by the host, including a region 5′ of the DNA todrive expression, and a 3′ region to terminate expression. The plasmidicvector harbouring the expression construct typically incorporates suchother functional components as an origin of replication, usuallyvirally-derived, to permit replication of the plasmid in the expressionhost, including bacterial hosts such as E. coli. To provide a markerenabling selection of stably transfected recombinant cells, the vectorwill also incorporate a gene conferring some survival advantage on thetransfectants, such as a gene coding for neomycin resistance in whichcase the transfectants are plated in medium with neomycin.

[0044] Included among the various recombinant DNA expression systemsthat can be used to achieve mammalian cell expression of DNA encodingNR3 or NR4 are those that exploit promoters of viruses that infectmammalian cells, such as the promoter from the cytomegalovirus (CMV),the Rous sarcoma virus (RSV), simian virus (SV40), murine mammary tumorvirus (MMTV) and others. Also useful to drive expression are promoterssuch as the long terminal repeat (LTR) of retroviruses, insect cellpromoters such as those regulated by temperature, and isolated fromDrosophila, as well as mammalian gene promoters such assteroid-inducible promoters and those regulated by heavy metals i.e. themetalothionein gene promoter. In order to achieve expression inbacterial hosts, such as E. coli, expression systems that exploit theexpression controlling regions of various E. coli and viral genes can beused to drive NR3/NR4 production including the lac gene, the trp gene,and regions of the lambda genome (P_(L) and P_(R)). Expression in yeastcan be achieved using the expression-controlling regions of genes suchas alcohol dehydrogenase and melibiase, and in Aspergillus, theexpression-controlling regions of genes such as alcohol dehydrogenaseand glucoamylase may be used. The expression controlling-regions ofbaculovirus may be used in the case of insect host cells.

[0045] For incorporation into the recombinant DNA expression vector, DNAcoding for the desired modulatory protein, e.g. an NR3 or NR4 protein,can be obtained by applying selected techniques of gene isolation orgene synthesis. As described in more detail in the examples herein, thepresent modulatory proteins, including naturally occurring variantsthereof, are encoded within the human genome, expressed in human braintissue, and can therefore be obtained by careful application ofconventional gene isolation and cloning techniques. This typically willentail extraction of total messenger RNA from a fresh source of humanbrain tissue, such as cerebellum, hippocampus or fetal brain tissue,followed by conversion of messenger RNA to cDNA and formation of alibrary in, for example, a bacterial plasmid, or more typically abacteriophage. Bacteriophage harbouring fragments of the human DNA aretypically grown by plating on a lawn of susceptible E. coli bacteria,such that individual phage plaques or colonies can be isolated. The DNAcarried by the phage colony is then typically immobilized on anitrocellulose or nylon-based hybridization membrane, and thenhybridized, under carefully controlled conditions, to a radioactively(or otherwise) labelled nucleotide probe of appropriate sequence toidentify the particular phage colony carrying NR3 or NR4-encoding DNA ofinterest. Typically, the gene or a portion thereof so identified issubcloned into a plasmidic vector for nucleic acid sequence analysis.

[0046] Having herein provided the nucleotide sequence of human NR3modulatory proteins, it will be appreciated that automated techniques ofgene synthesis and/or amplification can also be performed to generateDNA coding therefor. Because of the length of NR3-encoding DNA,application of automated synthesis may require staged gene construction,in which regions of the gene up to about 300 nucleotides in length aresynthesized individually and then ligated in correct succession forfinal assembly. Individually synthesized gene regions can be amplifiedprior to assembly using polymerase chain reaction (PCR) technology asgenerally described by Barnett et al. in Nucl. Acids Res. 18:3094, 1990.

[0047] The application of automated gene synthesis techniques providesan opportunity to generate sequence variants of naturally occurringmembers of the NR3 gene family. It will be appreciated, due to thedegeneracy associated with nucleotide triplet codons, that variantpolynucleotides coding for the NR3 receptors herein described can begenerated by substituting synonymous codons for those represented in thenaturally occurring polynucleotide sequences herein identified, such asthose identified in FIG. 1 and FIG. 3. For example, as would be known byone of skill in the art, arginine may be encoded by any one of sixcodons selected from CGA, CGC, CGG, CGU, AGA and AGG, threonine may beencoded by any one of four codons selected from ACA, ACC, ACG and ACU,while lysine is encoded by two codons, AAA and AAG. In addition,polynucleotides coding for synthetic variants of the NR3 receptors canbe generated which, for example, incorporate one or more, e.g. 1-10,single amino acid substitutions, deletions or additions. Since it willfor the most part be desirable to retain the modulatory activity of theNR3 protein for screening purposes, it is desirable to limit amino acidsubstitutions to those regions which are less critical for modulatoryactivity as may be elucidated upon domain mapping of the protein. Suchsubstitutions may include, for example, conservative amino acidsubstitutions such as isoleucine to leucine, or lysine to arginine.

[0048] With appropriate template DNA in hand, the technique of PCRamplification may also be used to directly generate all or part of thefinal gene. In this case, primers are synthesized which will prime thePCR amplification of the final product, either in one piece, or inseveral pieces that may be ligated together. This may be via step-wiseligation of blunt-ended, amplified DNA fragments, or preferentially viastep-wise ligation of fragments containing naturally occurringrestriction endonuclease sites. In this application, it is possible touse either cDNA or genomic DNA as the template for the PCRamplification. In the former case, the cDNA template can be obtainedfrom commercially available or self-constructed cDNA libraries ofvarious human brain tissues, including hippocampus and cerebellum.

[0049] Once obtained, the DNA encoding the desired modulatory protein isincorporated for expression into any suitable expression vector usingconventional procedures, and host cells are transfected therewith alsousing conventional procedures which include, for example, DNA-mediatedtransformation, electroporation, microinjection, or particle guntransformation. Expression vectors may be selected to providetransfected mammalian cell lines that express the modulatoryprotein-encoding DNA either transiently or in a stable manner. Fortransient expression, host cells are typically transfected with anexpression vector harbouring an origin of replication functional in amammalian cell. For stable expression, such replication origins areunnecessary, but the vectors will typically harbour a gene coding for aproduct that confers on the transfectants a survival advantage, toenable their selection. Genes coding for such selectable markersinclude, but are not limited to, the E. coli gpt gene which confersresistance to mycophenolic acid, the neo gene from transposon Tn5 whichconfers resistance to the antibiotic G418 and to neomycin, the dhfrsequence from murine cells or E. coli which changes the phenotype ofDHFR(−) cells into DHFR(+) cells, and the tk gene of herpes simplexvirus, which makes TK(−) cells phenotypically TK(+) cells. Bothtransient expression and stable expression can provide transfected celllines, and membrane preparations derived therefrom, for use in screeningassays.

[0050] The recombinant techniques described above can be equally appliedto EAA receptor production, in particular NMDA receptor production, asset out in the specific examples described herein and using, forexample, the DNA sequences provided in FIGS. 6 and 7, in the preparationof cells which heteromerically produce a modulatory protein and an NMDAreceptor. In this case, once the appropriate modulatory protein-encodingand NMDA receptor-encoding expression vectors have been prepared, thecells selected for expression are transfected with a mixture of theexpression vectors in the conventional manner.

[0051] For use in screening assays, cells transiently expressing theNR3/NR4-encoding DNA, and the NMDA receptor-encoding DNA, can be storedfrozen for later use, but because the rapid rate of plasmid replicationwill lead ultimately to cell death, usually in a few days, thetransfected cells should be used as soon as possible. Such assays may beperformed either with intact cells, or with membrane preparationsderived from such cells. The membrane preparations typically provide amore convenient substrate for the ligand screening experiments, and aretherefore preferred as substrates. To prepare membrane preparations forscreening purposes, i.e. ligand binding experiments, frozen intact cellsare homogenized while in cold water suspension and a membrane pellet iscollected after centrifugation. The pellet is re-suspended andre-centrifuged to remove endogenous ligands that would otherwise competefor binding in the assays. The membranes may then be used as such, orafter storage in lyophilized form, in the ligand binding assays.Alternatively, intact, fresh cells harvested about two days aftertransient transfection or after about the same period following freshplating of stably transfected cells, can be used for ligand bindingassays by the same methods as used for membrane preparations. When cellsare used, the cells must be harvested by more gentle centrifugation soas not to damage them, and all washing must be done in a bufferedmedium, for example in phosphate-buffered saline, to avoid osmotic shockand rupture of the cells.

[0052] The binding of a candidate ligand to a selected human modulatoryprotein of the invention, or a heteromeric receptor complex comprising amodulatory protein and an NMDA receptor, is evaluated typically using apredetermined amount of cell-derived membrane (measured for example byprotein determination), generally from about 25 ug to 100 ug.Competitive binding assays will be useful to evaluate the affinity of acandidate ligand for a heteromeric complex relative to glutamate. Thiscompetitive binding assay can be performed by incubating a membranepreparation with radiolabelled glutamate, for example [³H]-glutamate, inthe presence of unlabelled candidate ligand added at varyingconcentrations. Following incubation, either displaced or boundradiolabelled glutamate can be recovered and measured to determine therelative binding affinities of the candidate ligand and glutamate forthe particular receptor used as substrate. In this way, the affinitiesof various compounds for the heteromeric complex can be measured. Aswill be appreciated by one of skill in the art, binding assays such asradioimmunoassays and ELISA can also be used to determine bindingaffinity of a candidate ligand. Such competitive binding assays cannotbe used in the case of an NR3 or NR4 protein which is expressedhomomerically, in a state that does not naturally bind those ligandsbound by EAA receptors. Thus, the binding affinity of candidate ligandsfor the NR3 or NR4 proteins can be determined using a conventionalnon-competitive type binding assay. Those ligands determined to have anappropriate affinity for the homomeric modulatory protein, i.e. abinding affinity in the micromolar range, and more preferably in thenanomolar range, can then be selected to determine if their binding isspecific, and further, if their binding affects the pharmacological andfunctional characteristics of a heteromeric modulatory protein/receptorcomplex.

[0053] The NR3 and NR4 proteins of the present invention are functionalin a modulatory context, forming heteromeric receptor complexes,comprising a human modulatory protein and an EAA receptor, which exhibitelectrophysiological properties that are distinct from those exhibitedby either of the modulatory protein or the NMDA receptor components ofthe complex alone. The modulatory proteins are therefore useful, in theestablished manner, for screening candidate ligands for their ability tomodulate the ion channel activity of such receptor heteromericcomplexes. The present invention thus further provides, as a ligandscreening technique, a method of detecting interaction between acandidate ligand and a human modultory protein/receptor heteromericcomplex, comprising the steps of incubating the candidate ligand with acell that produces a human modulatory protein/receptor heteromericcomplex, or with a membrane preparation derived therefrom, and thenmeasuring the ligand-induced electrical current across said cell ormembrane.

[0054] As an alternative to using cells that express the modulatoryprotein, either homomerically or as a heteromeric receptor complex,ligand characterization may also be performed using cells (for exampleXenopus oocytes), that yield functional membrane-bound protein followingintroduction of messenger RNA coding for the NR3 or NR4 protein, in thecase of homomeric expression, or coding for a heteromeric complex, inthe case of heteromeric expression. Thus, NR3 or NR4 DNA is typicallysubcdoned into a plasmidic vector such that the introduced DNA may beeasily transcribed into RNA via an adjacent RNA transcription promotersupplied by the plasmidic vector, for example the T3 or T7 bacteriophagepromoters. RNA is then transcribed from the inserted gene in vitro, andisolated and purified therefrom for injection into Xenopus oocytes. Inthe case of a heteromeric complex, the RNA of the NMDA receptor formingthe complex is prepared in the same manner for injection into Xenopusoocytes simultaneously with RNA encoding the modulatory protein.Following the injection of nanoliter volumes of an RNA solution, theoocytes are left to incubate for up to several days, and are then testedfor the ability to respond to a particular ligand molecule supplied in abathing solution. In the heteromeric case, due to the fact that anactive membrane channel is formed through which ions may selectivelypass, the response of a particular ligand molecule in the bathingsolution may typically be measured as an electrical current utilizingmicroelectrodes inserted into the cell or placed on either side of acell-derived membrane preparation using the “patch-clamp” technique.

[0055] In addition to using DNA encoding the modulatory protein toconstruct cell lines useful for ligand screening, expression of the DNAcan, according to another aspect of the invention, be performed toproduce fragments of the protein in soluble form, for structureinvestigation, to raise antibodies and for other experimental uses. Itis therefore desirable in the first instance to facilitate thecharacterization of particular regions of NR3 or NR4 in quantity and inisolated form, i.e. free from the remainder of the full-length protein.One region of particular interest with regard to the modulatory functionof the NR3 and NR4 proteins is the extracellular N-terminal region. Toprepare a fragment of the N-terminal region, full-length DNA encodingthe modulatory protein may be modified by site-directed mutagenesis, tointroduce a translational stop codon into the extracellular N-terminalregion, immediately 5′ of the first transmembrane domain (TM1). Sincethere will no longer be produced any transmembrane domain(s) to “anchor”the protein into the membrane, expression of the modified cDNA willresult in the secretion, in soluble form, of only the extracellularN-terminal domain. Standard ligand-binding assays may then be performedto ascertain the degree of binding of a candidate compound to theextracellular domain so produced. Alternatively, a translational stopcodon may be introduced downstream of the first transmembrane domain toyield a fragment which retains the ability to anchor into the cellmembrane. In this way, a heteromeric channel comprising the N-terminalNR3/NR4 fragment can be formed and used to determine the extent ofmodulatory activity possessed by the fragment. It may of course benecessary, using site-directed mutagenesis, to produce differentversions of this extracellular region, or indeed any other extracellularregion of the protein, in order to map the modulatory domain withprecision.

[0056] Alternatively, it may be desirable to produce other regions ofthe modulatory protein, for example all or part of the carboxy-terminusthereof. In this case, site-directed mutagenesis and/or PCR-basedamplification techniques may readily be used to provide a definedfragment of the cDNA encoding the domain of interest. Once obtained,such DNA fragments can be expressed in the usual manner, eitherhomomerically to determine if the fragment has ligand-binding activity,or heteromerically to determine the extent to which the fragment retainsmodulatory activity. Conventional peptide synthetic techniques may alsobe used to make the desired C-terminal fragments or other fragments,e.g. a desired N-terminal fragment as noted above.

[0057] It will be appreciated that the production of such fragments maybe accomplished in a variety of host cells. Mammalian cells such as CHOcells may be used for this purpose, the expression typically beingdriven by an expression promoter capable of high-level expression, forexample, the CMV promoter. Alternately, non-mammalian cells, such asinsect Sf9 (Spodoptera frugiperda) cells may be used, with theexpression typically being driven by expression promoters of thebaculovirus, for example the strong, late polyhedrin protein promoter.Filamentous fungal expression systems may also be used to secrete largequantities of selected domains of the modulatory protein. Aspergillusnidulans for example, with the expression being driven by the alcApromoter, would constitute such an acceptable fungal expression system.In addition to such expression hosts, it will be further appreciatedthat any prokaryotic or other eukaryotic expression system capable ofexpressing heterologous genes or gene fragments, whether intracellularlyor extracellularly, would be similarly acceptable.

[0058] For use particularly in detecting the presence and/or location ofan NR3 or NR4 protein, for example in brain tissue, the presentinvention also provides, in another of its aspects, antibodies to theseproteins. Such antibodies will also have use as diagnostic agents, e.g.to determine if localized amounts or different forms of NR3 or NR4 inselected tissue types are indicative of a disease condition, and astherapeutic agents, by regulating the modulatory activity of an NR3 orNR4 protein on an NMDA receptor ion channel, to prevent diseaseconditions associated with overactive NMDA receptor ion channels.Preferably, for use therapeutically, the NR3 and NR4 antibodies employedare monoclonal antibodies.

[0059] To raise NR3 antibodies, for example, there may be used asimmunogen either the intact, soluble NR3 protein or an immunogenicfragment thereof, produced in a microbial or mammalian cell host asdescribed above or by standard peptide synthesis techniques. Regions ofthe NR3 protein particularly suitable for use as immunogenic fragmentsinclude those corresponding in sequence to an extracellular region ofthe receptor, or a portion of the extracellular region, such as peptidesconsisting of residues 27-557, or fragments thereof.

[0060] The raising of antibodies to the desired NR3 or NR4 protein orimmunogenic fragment can be achieved, for polyclonal antibodyproduction, using immunization protocols of conventional design, and anyof a variety of mammalian hosts, such as sheep, goats and rabbits. Formonoclonal antibody production, immunocytes such as splenocytes can berecovered from the immunized animal and fused, using hybridomatechnology, to myeloma cells. The fusion cell products, i.e. hybridomacells, are then screened by culturing in a selection medium, and cellsproducing the desired antibody are recovered for continuous growth, andantibody recovery. Recovered antibody can then be coupled covalently toa reporter molecule, i.e. a detectable label, such as a radiolabel,enzyme label, luminescent label or the like, optionally using linkertechnology established for this purpose.

[0061] In detectably labelled form, e.g. radiolabelled form,olignucleotides, including both DNA or RNA, coding for the human NR3 orNR4 modulatory protein and selected regions thereof, may also be used,in accordance with another aspect of the present invention, ashybridization probes for example to identify sequence-related genesresident in the human or other mammalian genomes (or cDNA libraries) orto locate DNA encoding an NR3 or NR4 protein in a specimen, such asbrain tissue. This can be done using either the intact coding region, ora fragment thereof, having radiolabelled nucleotides, for example,³²P-labelled nucleotides, incorporated therein. To identify theNR3-encoding DNA in a specimen, it is desirable to use either the fulllength cDNA coding therefor, or a fragment which is unique thereto. Withreference to FIG. 1 and the nucleotide numbering appearing thereon, suchnucleotide fragments include those comprising at least about 17 nucleicacids which correspond in sequence to an extracellular region of NR3DNA, e.g. the N-terminus thereof. Examples of suitable nucleotidefragments are the regions spanning nucleotides 8-1888 and 2732-5976 ofNR3-1. Such sequences, and the intact gene itself, may also be used ofcourse to clone NR3-related human genes, particularly cDNA equivalentsthereof, by standard hybridization techniques.

[0062] Embodiments of the present invention are described in detail inthe following specific Examples which should not be construed aslimiting.

EXAMPLE 1 Isolation of DNA Coding for Human NR3-1

[0063] A human NR2A DNA probe corresponding to a portion of nucleotidesequence of NR2A-1, namely the nucleotide region 1832-2361 (SEQ ID NO:9) as shown in FIG. 5, was generated by PCR-based amplification ofrecombinant bacteriophage lambda DNA isolated from an Eco RI-basedbacteriophage lambda library of human hippocampus cDNA (obtained fromStratagene Cloning Systems, La Jolla, Calif.). The following degenerateoligonucleotide primers were used in the PCR amplification: (SEQ IDNO:20) 1) 5′ GGGGTTTAGATCTGGGT-A/C/G/T-ATGATGTT-C/T-GT-A/ C/G/T-ATG 3′;and (SEQ ID NO:21) 2) 5′ GGGGTTTAGATCTGC-A/C/G/T-GC-A/G-TC-A/G-TA-A/G/T-AT-A/G-AA-A/G/C/T-GC 3′

[0064] The primers were used at a final concentration of 2 pmol/μl each,in a 50 μl reaction volume (10 mM Tris-HCl, pH 9.0; 50 mM KCl; 1.5 mMMgCl₂) containing 100 ng of recombinant human hippocampuscDNA/bacteriophage lambda DNA, 5 units of Thermus aquaticus DNApolymerase (Promega, Madison, Wis.) and 0.2 mM of eachdeoxyribonucleotide. Thirty-five cycles of amplification proceeded, withdenaturation at 95° C. for 1 min., annealing at 50° C. for 1 min., andprimer extension at 72° C. for 1 min., followed by a final cycle at 72°C. for 5 min. The 554 bp PCR product was purified from an agarose geland subcloned into the plasmid vector pT7Blue-T (Novagen, Madison, Wis.)for DNA sequencing.

[0065] A human NR4 DNA probe corresponding to a portion of nucleotidesequence of NR4, namely the nucleotide region 679-1263 as shown in FIG.4, was also generated as described above.

[0066] The 554 bp human NR2A and NR4 probes were radiolabelled with[α-³²P]dCTP using the Amersham Megaprime DNA labelling system (ArlingtonHeights, Ill.) to a specific activity of 1.0×10⁹ cpm/μg. The labelledprobes were used to screen approximately 1×10⁶ plaques of theEcoRI-based human hippocampus cDNA/bacteriophage lambda Zap II libraryidentified above and approximately 800,000 plaques of an Eco RI-basedhuman fetal brain cDNA/bacteriophage lambda Zap II library (obtainedfrom Stratagene). Fifteen positive plaques were identified on replicafilters under the following hybridization conditions: 6×SSPE, 50%formamide, 0.5% SDS, 100 μg/ml denatured salmon sperm DNA at 42° C. with1.85×10⁶ cpm probe per ml hybridization fluid. The filters were washedtwice with 2×SSPE, 0.5% SDS at 25° C. for 5 min., followed by a 15 min.wash at 42° C. The filters were exposed to X-ray film (Kodak, Rochester,N.Y.) overnight. The plaques were purified and excised as phagemidsaccording to the supplier's specifications, to generate aninsert-carrying Bluescript-SK variant of the phagemid vector.

[0067] DNA sequence analysis of the largest NR3 overlapping clones(isolated as pBS/FB2C and pBS/FB18) revealed a putative ATG initiationcodon together with about 210 nucleotides of 5′ untranslated (UTR)information and 4,452 nucleotides of amino acid coding information. Thisanalysis also revealed a termination codon as well as 1,307 nucleotidesof 3′ untranslated information. The entire DNA sequence of the NR3-1cDNA is provided in FIG. 1.

[0068] Partial DNA sequence analysis of the largest NR4 overlappingclones (isolated as pBS/H5A and pBS/H34A) indicated 1,785 nucleotides ofamino acid coding information. The DNA sequence of the partial NR4-1cDNA is provided in FIG. 4.

EXAMPLE 2 Isolation of DNA Coding for the Human NMDAR1-1 Receptor

[0069] A human NMDAR1 probe corresponding to a portion of nucleotidesequence of NMDAR1-1, namely the nucleotide region 2605-3213 as shown inFIG. 6, was generated by PCR-based amplification of recombinantbacteriophage lambda DNA isolated from an Eco RI-based bacteriophagelambda library of human hippocampus cDNA (obtained from StratageneCloning Systems, La Jolla, Calif.). The following degenerateoligonucleotide primers were used in the PCR amplification: (SEQ IDNO:22) 1) 5′ GGGGTTTGGATCCAA-A/G-GA-A/G-TGGAA-C/T-GGNATGA TG 3′; and(SEQ ID NO:23) 2) 5′ GGGGTTTAAGCTT-C/T-TC-G/A-TA-G/A-TT-G/A-TG-C/T-TT-C/T-TCCAT 3′

[0070] The primers were used at a final concentration of 5 pmol/μl each,in a 50 μl reaction volume (10 mM Tris-HCl, pH 9.0; 50 mM KCl; 1.5 mMMgCl₂) containing 100 ng of recombinant human hippocampuscDNA/bacteriophage lambda DNA, 5 units of Thermus aguaticus DNApolymerase (Promega, Madison, Wis.) and 0.2 mM of eachdeoxyribonucleotide. Thirty-five cycles of amplification proceeded, withdenaturation at 94° C. for 1 min., annealing at 51° C. for 1 min., andprimer extension at 72° C. for 1 min., followed by a final cycle at 72°C. for 5 min. The 674 bp PCR product was purified from an agarose geland subcloned into the plasmid vector pTZBlue-T (Novagen, Madison, Wis.)for DNA sequencing.

[0071] The 674 bp human NMDAR1 probe was radiolabelled with [α-³²P]dCTPusing the Amersham Megaprime DNA labelling system (Arlington Heights,Ill.) to a specific activity of 1.0-2.4×10⁹ cpm/ug. The labelled probewas used to screen approximately 400,000 plaques of an Eco RI-basedhuman hippocampus cDNA/bacteriophage lambda Zap II library. Thirty-fivepositive plaques were identified on replica filters under the followinghybridization conditions: 6×SSC, 50% formamide, 0.5% SDS, 100 ug/mldenatured salmon sperm DNA at 42° C. with 1.85×10⁶ cpm probe per mlhybridization fluid. The filters were washed with 2×SSC, 0.5% SDS at 25°C. for 5 min., followed by 15 min. washes at 37° C. and at 42° C. Thefilters were exposed to X-ray film (Kodak, Rochester, N.Y.) overnight.Twenty-eight plaques were purified and excised as phagemids according tothe supplier's specifications, to generate an insert-carryingBluescript-SK variant of the phagemid vector.

[0072] DNA sequence analysis of the clone NMDAR1-3C revealed 2,814nucleotides of amino acid coding information (938 amino acids). Theentire DNA sequence of the EcoRI-EcoRI NMDAR1-3C cDNA insert is providedherein by reference to the sequence of NMDAR1-1 set out in FIG. 6 and byreference to the sequence differences between NMDAR1-1 and NMDAR1-3Coutlined in FIG. 7. The NMDAR1-3C cDNA was subcloned into the pcDNA1-Ampmammalian expression vector (to form pcDNA1-Amp/hNR1-3C) using standardtechniques such as those described below in Example 3 for the subcloningof the NR2A clone into the pcDNA1-Amp vector.

[0073] It will be appreciated that the protocol described above can beused to isolate any of the NMDAR1 receptors in accordance with thepresent invention.

EXAMPLE 3 Construction of Genetically Engineered Cells Producing aHeteromeric Complex of Human NR3-1 and NMDAR1-3C

[0074] For transient expression in mammalian cells, cDNA coding forhuman NR3-1 was incorporated into the mammalian expression vectorpcDNA1-Amp (Invitrogen Corporation, San Diego, Calif.). This is amultifunctional 5 kbp plasmid vector designed for cDNA expression ineukaryotic systems, and cDNA analysis in prokaryotes. Incorporated onthe vector are the CMV immediate early gene promoter and enhancersequences, SV40 transcription termination and RNA processing signals,SV40 and polyoma virus origins of replication, M13 and ColE1 origins,Sp6 and T7 RNA promoters, and a gene conferring ampicillin resistance. Apolylinker is located. appropriately downstream of the CMV and T7promoters.

[0075] The strategy depicted in FIG. 2 was employed to facilitateincorporation of the NR3-1 cDNA into the expression vector. The FB2C 5′5.3 kbp BamHI/Sphl fragment was released from pBS/FB2C and ligated withthe 2.5 kbp BamHI/Sphl fragment of pBS/FB18(#20).Restriction-endonuclease digestion was performed to confirm properinsert orientation. The resulting plasmid was termed pBS/hNR3. The 4.8kbp EcoRI/NotI fragment of pBS/hNR3 was incorporated at the EcoRI/NotIsite in the pcDNA1-Amp polylinker. Restriction-endonuclease digestionand DNA sequence analysis was performed to confirm proper insertorientation. The resulting plasmid, designated pcDNA1-Amp/hNR3, was thenintroduced for transient expression into a selected mammalian cell host,in this case human embryonic kidney cells of the HEK293 lineage(available from the American Type Culture Collection, Rockville, Md.;ATCC CRL 1573).

[0076] The 7.8 kbp plasmid designated pBS/hNR3 carrying the NR3-1 DNA asa 4.8 kbp insert in a 3 kbp pBS plasmid background, was deposited, underthe terms of the Budapest Treaty, with the American Type CultureCollection in Rockville, Md., USA on Jun. 3, 1994 under accession numberATCC 75799.

[0077] For transient expression, HEK293 cells were transfected withapproximately 0.4 μg DNA (as pcDNA1-Amp/hNR3 or pcDNA1-Amp/hNR1-3C) per10⁵ HEK293 cells, by lipofectamine-mediated DNA transfection accordingto the manufacturer's (Life Technologies Inc., Gaithersburg, Md.)specifications. In coexpression experiments, i.e. for heteromericexpression of NR3-1 and NMDAR1-3C, the HEK293 cells were similarlytransfected with 0.8 μg of a DNA mixture containing pcDNA1-Amp/hNR3 andpcDNA1-Amp/hNR1-3C. Briefly, HEK293 cells were plated at a density of10⁵ cells/dish and then grown for 24 hours in 10% FBS-supplemented MEMmedium (Life Technologies Inc., Gaithersburg, Md.). The medium was thenremoved and cells were washed in OPTI-MEM I medium (Life TechnologiesInc.) lacking FBS, prior to transfection. A transfection solution (100μl) containing 2-4 μl of lipofectamine and DNA was then applied to thecells. After incubation for 4 hours at 37° C., cells were washed aspreviously described and then allowed to grow for 48 hours in 10%FBS-supplemented MEM medium containing 50 μM DL-AP5(2-amino-5-phosphonopentanoic acid) and 50 μM 7-chlorokyneurinic acidprior to electrophysiological recording.

[0078] In a like manner, stably transfected cell lines can also beprepared using various cell types as host: HEK293, CHO K1 or CHO Pro5.To construct these cell lines, cDNA coding for NR3-1 is incorporatedinto the mammalian expression vector pRc/CMV (Invitrogen Corp., SanDiego, Calif.) which enables stable expression. Insertion of the cDNAplaces it under the expression control of the CMV promoter and upstreamof the polyadenylation site and terminator of the bovine growth hormonegene, and into a vector background comprising the neomycin resistancegene (driven by the SV40 early promoter) as selectable marker. Tointroduce plasmids constructed as described above, the host cells arefirst seeded at a density of 5×10⁵ cells/dish in 10% FBS-supplementedMEM medium. After growth for 24 hours, fresh medium is added to theplates and three hours later, the cells are transfected using thelipofectin-mediated DNA transfection procedure according to themanufacturers specifications. Cells resistant to neomycin are selectedin 10% FBS-supplemented MEM medium containing G418 (1 mg/ml). Individualcolonies of G418-resistant cells are isolated about 2-3 weeks later,clonally selected and then propagated for assay purposes.

EXAMPLE 4 Electrophysiological Characterization

[0079] Standard whole-cell voltage-clamp (Axopatch 1B, Axon Instruments,Foster City, Calif.) techniques were used to record 60 μM NMDA-evokedcurrents in HEK293 cells transiently transfected as described in Example3 and expressing hNR3-1 heteromerically with the NMDAR1-3C receptor. Thecells were rinsed prior to recording with a solution of 130 mM NaCl, 5.4mM KCl, 1.8 mM CaCl₂, 10 μM glycine, 5 mM HEPES, pH 7.2 (300 mOsm.).Single electrode, voltage-clamp recordings were carried out usingthin-walled borosilicate glass electrodes (WPI-TW150-F4, WPI Inc.,Sarasota, Fla.) filled with an intracellular solution of 140 mM CsCl, 1mM MgCl₂, 10 mM EGTA, 10 mM HEPES, pH 7.2 (adjusted with 1 M CsOH). NMDAapplication using a computer controlled array of perfusion barrelsallowed for fast application and continuous perfusion with control or 1mM Mg²⁺-containing solutions (lag<50 milliseconds).

[0080] The results of the electrophysiological characterization aredepicted in FIG. 10. Points at which NMDA was applied are indicated withblack bars above the recordings. No NMDA-induced depolarizations wereobserved in HEK293 cells transiently transfected with NMDAR1-3C alone,or with NR3 alone. NMDA-induced depolarizations were, however, observedin HEK293 cells transiently transfected with both NR3-1 and NMDAR1-3C.As illustrated, these latter currents were blocked by 1 mM MgCl₂, aresult which is characteristic of NMDA-gated ion channels.

[0081] This electrophysiological characterization indicates that theNR3/NMDA receptor heteromeric complex functions in an authentic manner,and can therefore be used to reliably predict the functional “signature”of its non-recombinant counterpart from intact human brain. Thesefeatures make the recombinant receptor especially useful for selectingand characterizing ligand compounds which bind to or otherwise modulatethe receptor, and/or for selecting and characterizing compounds whichmay act by displacing other ligands from the receptor. The isolation ofthe NR3 protein in a pure form, and its expression with an NMDA receptoras a single, homogenous complex, therefore frees theelectrophysiological assay from the lack of precision introduced whencomplex receptor preparations from human and non-human brains are usedto attempt such characterizations.

[0082] It will be appreciated that the protocol described above can beused to determine the electrophysiological characteristics of otherNR3INMDA heteromeric receptor complexes, such as for example, theNR3-2/NMDAR1-1 complex.

EXAMPLE 5 Ligand-binding Assays on Heteromeric NR3-1/NMDAR1-3C Complex

[0083] Frozen transfected cells, prepared as described in Example 3above, are resuspended in ice-cold distilled water, sonicated for 5seconds, and centrifuged for 10 minutes at 50,000×g. The supernatant isdiscarded and the membrane pellet is stored frozen at −70° C.

[0084] Cell membrane pellets are resuspended in ice cold 50 mM Tris-HCl,pH 7.55, and centrifuged again at 50,000×g for 10 minutes in order toremove endogenous glutamate that would otherwise compete for binding.The pellets are resuspended in ice cold 50 mM Tris-HCl, pH 7.55, andused for the binding experiments described below. Protein concentrationsare determined using the Pierce reagent with BSA as an internalstandard.

[0085] Binding assays are performed using a 25-100 μg protein equivalentof the cell membrane preparation, and a selected radiolabeled ligand. Inparticular, for MK-801-binding assays, incubation mixtures consist of 20nM (+)-[3-³H]MK-801 (30 Ci/mmole), 20 μM glycine, and 1 mM L-glutamatein cold incubation buffer (50 mM Tris-HCl, pH 7.55) at a final volume of250 μl. Non-specific binding is determined in the presence of 1 mM(+)MK-801. For glutamate binding assays, incubation mixtures consist of30 nM [3,4-³H]-L-glutamate (47.3 Ci/mmole) in cold incubation buffer ata final volume of 250 μl. Non-specific binding is determined in thepresence of 1 mM L-glutamate and displacement is determined in thepresence of 1 mM NMDA, 1 mM kainate, or 1 mM AMPA. The reaction mixturesare incubated on ice for 60 minutes in plastic mini-vials. Bound andfree ligand are separated by centrifugation for 30 minutes at 50,000×g.The pellets are washed three times in 4 ml of the cold incubationbuffer, and then 4 ml of Beckman Ready-Protein Plus scintillationcocktail was added for liquid scintillation counting.

[0086] It will be appreciated that the protocol described above can beused to determine the pharmacological characteristics of other NR3/NMDAheteromeric receptor complexes, such as for example, the NR3-1/NMDAR1-1complex.

EXAMPLE 6 Ligand-binding Assay for the Homomeric Expression of NR3-1

[0087] Frozen transfected cells, prepared as described in Example 3above and expressing NR3-1 in the absence of an NMDA receptor, areresuspended in ice-cold distilled water, sonicated for 5 seconds, andcentrifuged for 10 minutes at 50,000×g. The supernatant is discarded andthe membrane pellet is stored frozen at −70° C.

[0088] Cell membrane pellets are resuspended in ice cold 50 mM Tris-HCl,pH 7.55, and centrifuged again at 50,000×g for 10 minutes in order toremove endogenous ligands that might otherwise compete for binding. Thepellets are resuspended in ice cold 50 mM Tris-HCl, pH 7.55, and usedfor the binding experiments described below. Protein concentrations aredetermined using the Pierce reagent with BSA as an internal standard.

[0089] Binding assays are performed using a 25-100 μg protein equivalentof the cell membrane preparation, and a selected radiolabeled ligand incold incubation buffer (50 mM Tris-HCl, pH 7.55) at a final volume of250 μl. Non-specific binding is determined in the presence of theunlabeled ligand. The reaction mixtures are incubated on ice for 60minutes in plastic mini-vials. Bound and free ligand are, separated bycentrifugation for 30 minutes at 50,000×g. The pellets are washed threetimes in 4 ml of the cold incubation buffer, and then 4 ml of BeckmanReady-Protein Plus scintillation cocktail are added for liquidscintillation counting.

[0090] Having determined that the selected ligand binds specifically toNR3-1, i.e. that unlabelled ligand competes for binding with thelabelled form of that ligand, and that the binding is saturable, theligand is then tested for its ability to affect the heteromericexpression of NR3-1, i.e. when coexpressed with an NMDA receptor asdescribed above. Appropriate experiments for this purpose include theligand binding experiment described in Example 5, and theelectrophysiological characterization described in Example 4.

EXAMPLE 7 Isolation and Cloning of the NR3-2 Variant

[0091] The procedures described in Examples 1 and 3 for isolating andcloning the NR3-1 protein are applied equally for the isolation andcloning of NR3-2 and other naturally occuring variants of NR3-1,particularly in view of the high sequence homology between the NR3-1receptor and the NR3-2 variant.

[0092] Moreover, the electrophysiological and ligand-binding assaysdescribed in Examples 4, 5 and 6, respectively, are used in the mannerdescribed to determine the electrophysiological and ligand bindingcharacteristics of NR3-2 and other NR3-1 variants.

1 23 5983 base pairs nucleic acid double linear CDS 218..4669 1GAATTCCTTT GAATTTGCAT CTCTTCAAGA CACAAGATTA AAACAAAATT TACGCTAAAT 60TGGATTTTAA ATTATCTTCC GTTCATTTAT CCTTCGTCTT TCTTATGTGG ATATGCAAGC 120GAGAAGAAGG GACTGGACAT TCCCAACATG CTCACTCCCT TAATCTGTCC GTCTAGAGGT 180TTGGCTTCTA CAAACCAAGG GAGTCGACGA GTTGAAG ATG AAG CCC AGA GCG GAG 235 MetLys Pro Arg Ala Glu 1 5 TGC TGT TCT CCC AAG TTC TGG TTG GTG TTG GCC GTCCTG GCC GTG TCA 283 Cys Cys Ser Pro Lys Phe Trp Leu Val Leu Ala Val LeuAla Val Ser 10 15 20 GGC AGC AGA GCT CGT TCT CAG AAG AGC CCC CCC AGC ATTGGC ATT GCT 331 Gly Ser Arg Ala Arg Ser Gln Lys Ser Pro Pro Ser Ile GlyIle Ala 25 30 35 GTC ATC CTC GTG GGC ACT TCC GAC GAG GTG GCC ATC AAG GATGCC CAC 379 Val Ile Leu Val Gly Thr Ser Asp Glu Val Ala Ile Lys Asp AlaHis 40 45 50 GAG AAA GAT GAT TTC CAC CAT CTC TCC GTG GTA CCC CGG GTG GAACTG 427 Glu Lys Asp Asp Phe His His Leu Ser Val Val Pro Arg Val Glu Leu55 60 65 70 GTA GCC ATG AAT GAG ACC GAC CCA AAG AGC ATC ATC ACC CGC ATCTGT 475 Val Ala Met Asn Glu Thr Asp Pro Lys Ser Ile Ile Thr Arg Ile Cys75 80 85 GAT CTC ATG TCT GAC CGG AAG ATC CAG GGG GTG GTG TTT GCT GAT GAC523 Asp Leu Met Ser Asp Arg Lys Ile Gln Gly Val Val Phe Ala Asp Asp 9095 100 ACA GAC CAG GAA GCC ATC GCC CAG ATC CTC GAT TTC ATT TCA GCA CAG571 Thr Asp Gln Glu Ala Ile Ala Gln Ile Leu Asp Phe Ile Ser Ala Gln 105110 115 ACT CTC ACC CCG ATC CTG GGC ATC CAC GGG GGC TCC TCT ATG ATA ATG619 Thr Leu Thr Pro Ile Leu Gly Ile His Gly Gly Ser Ser Met Ile Met 120125 130 GCA GAT AAG GAT GAA TCC TCC ATG TTC TTC CAG TTT GGC CCA TCA ATT667 Ala Asp Lys Asp Glu Ser Ser Met Phe Phe Gln Phe Gly Pro Ser Ile 135140 145 150 GAA CAG CAA GCT TCC GTA ATG CTC AAC ATC ATG GAA GAA TAT GACTGG 715 Glu Gln Gln Ala Ser Val Met Leu Asn Ile Met Glu Glu Tyr Asp Trp155 160 165 TAC ATC TTT TCT ATC GTC ACC ACC TAT TTC CCT GGC TAC CAG GACTTT 763 Tyr Ile Phe Ser Ile Val Thr Thr Tyr Phe Pro Gly Tyr Gln Asp Phe170 175 180 GTA AAC AAG ATC CGC AGC ACC ATT GAG AAT AGC TTT GTG GGC TGGGAG 811 Val Asn Lys Ile Arg Ser Thr Ile Glu Asn Ser Phe Val Gly Trp Glu185 190 195 CTA GAG GAG GTC CTC CTA CTG GAC ATG TCC CTG GAC GAT GGA GATTCT 859 Leu Glu Glu Val Leu Leu Leu Asp Met Ser Leu Asp Asp Gly Asp Ser200 205 210 AAG ATC CAG AAT CAG CTC AAG AAA CTT CAA AGC CCC ATC ATT CTTCTT 907 Lys Ile Gln Asn Gln Leu Lys Lys Leu Gln Ser Pro Ile Ile Leu Leu215 220 225 230 TAC TGT ACC AAG GAA GAA GCC ACC TAC ATC TTT GAA GTG GCCAAC TCA 955 Tyr Cys Thr Lys Glu Glu Ala Thr Tyr Ile Phe Glu Val Ala AsnSer 235 240 245 GTA GGG CTG ACT GGC TAT GGC TAC ACG TGG ATC GTG CCC AGTCTG GTG 1003 Val Gly Leu Thr Gly Tyr Gly Tyr Thr Trp Ile Val Pro Ser LeuVal 250 255 260 GCA GGG GAT ACA GAC ACA GTG CCT GCG GAG TTC CCC ACT GGGCTC ATC 1051 Ala Gly Asp Thr Asp Thr Val Pro Ala Glu Phe Pro Thr Gly LeuIle 265 270 275 TCT GTA TCA TAT GAT GAA TGG GAC TAT GGC CTC CCC GCC AGAGTG AGA 1099 Ser Val Ser Tyr Asp Glu Trp Asp Tyr Gly Leu Pro Ala Arg ValArg 280 285 290 GAT GGA ATT GCC ATA ATC ACC ACT GCT GCT TCT GAC ATG CTGTCT GAG 1147 Asp Gly Ile Ala Ile Ile Thr Thr Ala Ala Ser Asp Met Leu SerGlu 295 300 305 310 CAC AGC TTC ATC CCT GAG CCC AAA AGC AGT TGT TAC AACACC CAC GAG 1195 His Ser Phe Ile Pro Glu Pro Lys Ser Ser Cys Tyr Asn ThrHis Glu 315 320 325 AAG AGA ATC TAC CAG TCC AAT ATG CTA AAT AGG TAT CTGATC AAT GTC 1243 Lys Arg Ile Tyr Gln Ser Asn Met Leu Asn Arg Tyr Leu IleAsn Val 330 335 340 ACT TTT GAG GGG AGG AAT TTG TCC TTC AGT GAA GAT GGCTAC CAG ATG 1291 Thr Phe Glu Gly Arg Asn Leu Ser Phe Ser Glu Asp Gly TyrGln Met 345 350 355 CAC CCG AAA CTG GTG ATA ATT CTT CTG AAC AAG GAG AGGAAG TGG GAA 1339 His Pro Lys Leu Val Ile Ile Leu Leu Asn Lys Glu Arg LysTrp Glu 360 365 370 AGG GTG GGG AAG TGG AAA GAC AAG TCC CTG CAG ATG AAGTAC TAT GTG 1387 Arg Val Gly Lys Trp Lys Asp Lys Ser Leu Gln Met Lys TyrTyr Val 375 380 385 390 TGG CCC CGA ATG TGT CCA GAG ACT GAA GAG CAG GAGGAT GAC CAT CTG 1435 Trp Pro Arg Met Cys Pro Glu Thr Glu Glu Gln Glu AspAsp His Leu 395 400 405 AGC ATT GTG ACC CTG GAG GAG GCA CCA TTT GTC ATTGTG GAA AGT GTG 1483 Ser Ile Val Thr Leu Glu Glu Ala Pro Phe Val Ile ValGlu Ser Val 410 415 420 GAC CCT CTG AGT GGA ACC TGC ATG AGG AAC ACA GTCCCC TGC CAA AAA 1531 Asp Pro Leu Ser Gly Thr Cys Met Arg Asn Thr Val ProCys Gln Lys 425 430 435 CGC ATA GTC ACT GAG AAT AAA ACA GAC GAG GAG CCGGGT TAC ATC AAA 1579 Arg Ile Val Thr Glu Asn Lys Thr Asp Glu Glu Pro GlyTyr Ile Lys 440 445 450 AAA TGC TGC AAG GGG TTC TGT ATT GAC ATC CTT AAGAAA ATT TCT AAA 1627 Lys Cys Cys Lys Gly Phe Cys Ile Asp Ile Leu Lys LysIle Ser Lys 455 460 465 470 TCT GTG AAG TTC ACC TAT GAC CTT TAC CTG GTTACC AAT GGC AAG CAT 1675 Ser Val Lys Phe Thr Tyr Asp Leu Tyr Leu Val ThrAsn Gly Lys His 475 480 485 GGG AAG AAA ATC AAT GGA ACC TGG AAT GGT ATGATT GGA GAG GTG GTC 1723 Gly Lys Lys Ile Asn Gly Thr Trp Asn Gly Met IleGly Glu Val Val 490 495 500 ATG AAG AGG GCC TAC ATG GCA GTG GGC TCA CTCACC ATC AAT GAG GAA 1771 Met Lys Arg Ala Tyr Met Ala Val Gly Ser Leu ThrIle Asn Glu Glu 505 510 515 CGA TCG GAG GTG GTC GAC TTC TCT GTG CCC TTCATA GAG ACA GGC ATC 1819 Arg Ser Glu Val Val Asp Phe Ser Val Pro Phe IleGlu Thr Gly Ile 520 525 530 AGT GTC ATG GTG TCA CGC AGC AAT GGG ACT GTCTCA CCT TCT GCC TTC 1867 Ser Val Met Val Ser Arg Ser Asn Gly Thr Val SerPro Ser Ala Phe 535 540 545 550 TTA GAG CCA TTC AGC GCT GAC GTA TGG GTGATG ATG TTT GTG ATG CTG 1915 Leu Glu Pro Phe Ser Ala Asp Val Trp Val MetMet Phe Val Met Leu 555 560 565 CTC ATC GTC TCA GCC GTG GCT GTC TTT GTCTTT GAG TAC TTC AGC CCT 1963 Leu Ile Val Ser Ala Val Ala Val Phe Val PheGlu Tyr Phe Ser Pro 570 575 580 GTG GGT TAT AAC AGG TGC CTC GCT GAT GGCAGA GAG CCT GGT GGA CCC 2011 Val Gly Tyr Asn Arg Cys Leu Ala Asp Gly ArgGlu Pro Gly Gly Pro 585 590 595 TCT TTC ACC ATC GGC AAA GCT ATT TGG TTGCTC TGG GGT CTG GTG TTT 2059 Ser Phe Thr Ile Gly Lys Ala Ile Trp Leu LeuTrp Gly Leu Val Phe 600 605 610 AAC AAC TCC GTA CCT GTG CAG AAC CCA AAGGGG ACC ACC TCC AAG ATC 2107 Asn Asn Ser Val Pro Val Gln Asn Pro Lys GlyThr Thr Ser Lys Ile 615 620 625 630 ATG GTG TCA GTG TGG GCC TTC TTT GCTGTC ATC TTC CTG GCC AGC TAC 2155 Met Val Ser Val Trp Ala Phe Phe Ala ValIle Phe Leu Ala Ser Tyr 635 640 645 ACT GCC AAC TTA GCT GCC TTC ATG ATCCAA GAG GAA TAT GTG GAC CAG 2203 Thr Ala Asn Leu Ala Ala Phe Met Ile GlnGlu Glu Tyr Val Asp Gln 650 655 660 GTT TCT GGC CTG AGC GAC AAA AAG TTCCAG AGA CCT AAT GAC TTC TCA 2251 Val Ser Gly Leu Ser Asp Lys Lys Phe GlnArg Pro Asn Asp Phe Ser 665 670 675 CCC CCT TTC CGC TTT GGG ACC GTG CCCAAC GGC AGC ACA GAG AGA AAT 2299 Pro Pro Phe Arg Phe Gly Thr Val Pro AsnGly Ser Thr Glu Arg Asn 680 685 690 ATT CGC AAT AAC TAT GCA GAA ATG CATGCC TAC ATG GGA AAG TTC AAC 2347 Ile Arg Asn Asn Tyr Ala Glu Met His AlaTyr Met Gly Lys Phe Asn 695 700 705 710 CAG AGG GGT GTA GAT GAT GCA TTGCTC TCC CTG AAA ACA GGG AAA CTG 2395 Gln Arg Gly Val Asp Asp Ala Leu LeuSer Leu Lys Thr Gly Lys Leu 715 720 725 GAT GCC TTC ATC TAT GAT GCA GCAGTG CTG AAC TAT ATG GCA GGC AGA 2443 Asp Ala Phe Ile Tyr Asp Ala Ala ValLeu Asn Tyr Met Ala Gly Arg 730 735 740 GAT GAA GGC TGC AAG CTG GTG ACCATT GGC AGT GGG AAG GTC TTT GCT 2491 Asp Glu Gly Cys Lys Leu Val Thr IleGly Ser Gly Lys Val Phe Ala 745 750 755 TCC ACT GGC TAT GGC ATT GCC ATCCAA AAA GAT TCT GGG TGG AAG CGC 2539 Ser Thr Gly Tyr Gly Ile Ala Ile GlnLys Asp Ser Gly Trp Lys Arg 760 765 770 CAG GTG GAC CTT GCT ATC CTG CAGCTC TTT GGA GAT GGG GAG ATG GAA 2587 Gln Val Asp Leu Ala Ile Leu Gln LeuPhe Gly Asp Gly Glu Met Glu 775 780 785 790 GAA CTG GAA GCT CTC TGG CTCACT GGC ATT TGT CAC AAT GAG AAG AAT 2635 Glu Leu Glu Ala Leu Trp Leu ThrGly Ile Cys His Asn Glu Lys Asn 795 800 805 GAG GTC ATG AGC AGC CAG CTGGAC ATT GAC AAC ATG GCA GGG GTC TTC 2683 Glu Val Met Ser Ser Gln Leu AspIle Asp Asn Met Ala Gly Val Phe 810 815 820 TAC ATG TTG GGG GCG GCC ATGGCT CTC AGC CTC ATC ACC TTC ATC TGC 2731 Tyr Met Leu Gly Ala Ala Met AlaLeu Ser Leu Ile Thr Phe Ile Cys 825 830 835 GAA CAC CTT TTC TAT TGG CAGTTC CGA CAT TGC TTT ATG GGT GTC TGT 2779 Glu His Leu Phe Tyr Trp Gln PheArg His Cys Phe Met Gly Val Cys 840 845 850 TCT GGC AAG CCT GGC ATG GTCTTC TCC ATC AGC AGA GGT ATC TAC AGC 2827 Ser Gly Lys Pro Gly Met Val PheSer Ile Ser Arg Gly Ile Tyr Ser 855 860 865 870 TGC ATC CAT GGG GTG GCGATC GAG GAG CGC CAG TCT GTA ATG AAC TCC 2875 Cys Ile His Gly Val Ala IleGlu Glu Arg Gln Ser Val Met Asn Ser 875 880 885 CCC ACT GCA ACC ATG AACAAC ACA CAC TCC AAC ATC CTG CGC CTG CTG 2923 Pro Thr Ala Thr Met Asn AsnThr His Ser Asn Ile Leu Arg Leu Leu 890 895 900 CGC ACG GCC AAG AAC ATGGCT AAC CTG TCT GGT GTG AAT GGC TCA CCG 2971 Arg Thr Ala Lys Asn Met AlaAsn Leu Ser Gly Val Asn Gly Ser Pro 905 910 915 CAG AGG CCC CTG GAC TTCATC CGA CGG GAG TCA TCC GTC TAT GAC ATC 3019 Gln Arg Pro Leu Asp Phe IleArg Arg Glu Ser Ser Val Tyr Asp Ile 920 925 930 TCA GAG CAC CGC CGC AGCTTC ACG CAT TCT GAC TGC AAA TCC TAC AAC 3067 Ser Glu His Arg Arg Ser PheThr His Ser Asp Cys Lys Ser Tyr Asn 935 940 945 950 AAC CCG CCC TGT GAGGAG AAC CTC TTC AGT GAC TAC ATC AGT GAG GTA 3115 Asn Pro Pro Cys Glu GluAsn Leu Phe Ser Asp Tyr Ile Ser Glu Val 955 960 965 GAG AGA ACG TTC GGGAAC CTG CAG CTG AAG GAC AGC AAC GTG TAC CAA 3163 Glu Arg Thr Phe Gly AsnLeu Gln Leu Lys Asp Ser Asn Val Tyr Gln 970 975 980 GAT CAC TAC CAC CATCAC CAC CGG CCC CAT AGT ATT GGC AGT GCC AGC 3211 Asp His Tyr His His HisHis Arg Pro His Ser Ile Gly Ser Ala Ser 985 990 995 TCC ATC GAT GGG CTCTAC GAC TGT GAC AAC CCA CCC TTC ACC ACC CAG 3259 Ser Ile Asp Gly Leu TyrAsp Cys Asp Asn Pro Pro Phe Thr Thr Gln 1000 1005 1010 TCC AGG TCC ATCAGC AAG AAG CCC CTG GAC ATC GGC CTC CCC TCC TCC 3307 Ser Arg Ser Ile SerLys Lys Pro Leu Asp Ile Gly Leu Pro Ser Ser 1015 1020 1025 1030 AAG CACAGC CAG CTC AGT GAC CTG TAC GGC AAA TTC TCC TTC AAG AGC 3355 Lys His SerGln Leu Ser Asp Leu Tyr Gly Lys Phe Ser Phe Lys Ser 1035 1040 1045 GACCGC TAC AGT GGC CAC GAC GAC TTG ATC CGC TCC GAT GTC TCT GAC 3403 Asp ArgTyr Ser Gly His Asp Asp Leu Ile Arg Ser Asp Val Ser Asp 1050 1055 1060ATC TCA ACC CAC ACC GTC ACC TAT GGG AAC ATC GAG GGC AAT GCC GCC 3451 IleSer Thr His Thr Val Thr Tyr Gly Asn Ile Glu Gly Asn Ala Ala 1065 10701075 AAG AGG CGT AAG CAG CAA TAT AAG GAC AGC CTG AAG AAG CGG CCT GCC3499 Lys Arg Arg Lys Gln Gln Tyr Lys Asp Ser Leu Lys Lys Arg Pro Ala1080 1085 1090 TCG GCC AAG TCC CGC AGG GAG TTT GAC GAG ATC GAG CTG GCCTAC CGT 3547 Ser Ala Lys Ser Arg Arg Glu Phe Asp Glu Ile Glu Leu Ala TyrArg 1095 1100 1105 1110 CGC CGA CCG CCC CGC TCC CCT GAC CAC AAG CGC TACTTC AGG GAC AAG 3595 Arg Arg Pro Pro Arg Ser Pro Asp His Lys Arg Tyr PheArg Asp Lys 1115 1120 1125 GAA GGG CTA CGG GAC TTC TAC CTG GAC CAG TTCCGA ACA AAG GAG AAC 3643 Glu Gly Leu Arg Asp Phe Tyr Leu Asp Gln Phe ArgThr Lys Glu Asn 1130 1135 1140 TCA CCC CAC TGG GAG CAC GTA GAC CTG ACCGAC ATC TAC AAG GAG CGG 3691 Ser Pro His Trp Glu His Val Asp Leu Thr AspIle Tyr Lys Glu Arg 1145 1150 1155 AGT GAT GAC TTT AAG CGC GAC TCC GTCAGC GGA GGA GGG CCC TGT ACC 3739 Ser Asp Asp Phe Lys Arg Asp Ser Val SerGly Gly Gly Pro Cys Thr 1160 1165 1170 AAC AGG TCT CAC ATC AAG CAC GGGACG GGC GAC AAA CAC GGC GTG GTC 3787 Asn Arg Ser His Ile Lys His Gly ThrGly Asp Lys His Gly Val Val 1175 1180 1185 1190 AGC GGG GTA CCT GCA CCTTGG GAG AAG AAC CTG ACC AAC GTG GAG TGG 3835 Ser Gly Val Pro Ala Pro TrpGlu Lys Asn Leu Thr Asn Val Glu Trp 1195 1200 1205 GAG GAC CGG TCC GGGGGC AAC TTC TGC CGC AGC TGT CCC TCC AAG CTG 3883 Glu Asp Arg Ser Gly GlyAsn Phe Cys Arg Ser Cys Pro Ser Lys Leu 1210 1215 1220 CAC AAC TAC TCCACG ACG GTG ACG GGT CAG AAC TCG GGC AGG CAG GCG 3931 His Asn Tyr Ser ThrThr Val Thr Gly Gln Asn Ser Gly Arg Gln Ala 1225 1230 1235 TGC ATC CGGTGT GAG GCT TGC AAG AAA GCA GGC AAC CTG TAT GAC ATC 3979 Cys Ile Arg CysGlu Ala Cys Lys Lys Ala Gly Asn Leu Tyr Asp Ile 1240 1245 1250 AGT GAGGAC AAC TCC CTG CAG GAA CTG GAC CAG CCG GCT GCC CCA GTG 4027 Ser Glu AspAsn Ser Leu Gln Glu Leu Asp Gln Pro Ala Ala Pro Val 1255 1260 1265 1270GCG GTG ACG TCA AAC GCC TCC ACC ACT AAG TAC CCT CAG AGC CCG ACT 4075 AlaVal Thr Ser Asn Ala Ser Thr Thr Lys Tyr Pro Gln Ser Pro Thr 1275 12801285 AAT TCC AAG GCC CAG AAG AAG AAC CGG AAC AAA CTG CGC CGG CAG CAC4123 Asn Ser Lys Ala Gln Lys Lys Asn Arg Asn Lys Leu Arg Arg Gln His1290 1295 1300 TCC TAC GAC ACC TTC GTG GAC CTG CAG AAG GAA GAA GCC GCCCTG GCC 4171 Ser Tyr Asp Thr Phe Val Asp Leu Gln Lys Glu Glu Ala Ala LeuAla 1305 1310 1315 CCG CGC AGC GTA AGC CTG AAA GAC AAG GGC CGA TTC ATGGAT GGG AGC 4219 Pro Arg Ser Val Ser Leu Lys Asp Lys Gly Arg Phe Met AspGly Ser 1320 1325 1330 CCC TAC GCC CAC ATG TTT GAG ATG TCA GCT GGC GAGAGC ACC TTT GCC 4267 Pro Tyr Ala His Met Phe Glu Met Ser Ala Gly Glu SerThr Phe Ala 1335 1340 1345 1350 AAC AAC AAG TCC TCA GTG CCC ACT GCC GGACAT CAC CAC CAC AAC AAC 4315 Asn Asn Lys Ser Ser Val Pro Thr Ala Gly HisHis His His Asn Asn 1355 1360 1365 CCC GGC GGC GGG TAC ATG CTC AGC AAGTCG CTC TAC CCT GAC CGG GTC 4363 Pro Gly Gly Gly Tyr Met Leu Ser Lys SerLeu Tyr Pro Asp Arg Val 1370 1375 1380 ACG CAA AAC CCT TTC ATC CCC ACTTTT GGG GAC GAC CAG TGC TTG CTC 4411 Thr Gln Asn Pro Phe Ile Pro Thr PheGly Asp Asp Gln Cys Leu Leu 1385 1390 1395 CAT GGC AGC AAA TCC TAC TTCTTC AGG CAG CCC ACG GTG GCG GGG GCG 4459 His Gly Ser Lys Ser Tyr Phe PheArg Gln Pro Thr Val Ala Gly Ala 1400 1405 1410 TCG AAA GCC AGG CCG GACTTC CGG GCC CTT GTC ACC AAC AAG CCG GTG 4507 Ser Lys Ala Arg Pro Asp PheArg Ala Leu Val Thr Asn Lys Pro Val 1415 1420 1425 1430 GTC TCG GCC CTTCAT GGG GCC GTG CCA GCC CGT TTC CAG AAG GAC ATC 4555 Val Ser Ala Leu HisGly Ala Val Pro Ala Arg Phe Gln Lys Asp Ile 1435 1440 1445 TGT ATA GGGAAC CAG TCC AAC CCC TGT GTG CCT AAC AAC AAA AAC CCC 4603 Cys Ile Gly AsnGln Ser Asn Pro Cys Val Pro Asn Asn Lys Asn Pro 1450 1455 1460 AGG GCTTTC AAT GGC TCC AGC AAT GGG CAT GTT TAT GAG AAA CTT TCT 4651 Arg Ala PheAsn Gly Ser Ser Asn Gly His Val Tyr Glu Lys Leu Ser 1465 1470 1475 AGTATT GAG TCT GAT GTC TGAGTGAGGG AACAGAGAGG TTAAGGTGGG 4699 Ser Ile GluSer Asp Val 1480 TACGGGAGGG TAAGGCTGTG GGTCGCGTGA TGCGCATGTC ACGGAGGGTGACGGGGGTGA 4759 ACTTGGTTCC CATTTGCTCC TTTCTTGTTT TAATTTATTT ATGGGGATCCTGGAGTTCTG 4819 GTTCCTACTG GGGGCAACCC TGGTGACCAG CACCATCTCT CCTCCTTTTCACAGTTCTCT 4879 CCTTCTTCCC CCCGCTGTCA GCCATTCCTG TTCCCATGAG ATGATGCCATGGGTCTCAGC 4939 AGGGGAGGGT AGAGCGGAGA AAGGAAGGGC AGCATGCGGG CTTCCTCCTGGTGTGGAAGA 4999 GCTCCTTGAT ATCCTCTTTG AGTGAAGCTG GGAGAACCAA AAAGAGGCTATGTGAGCACA 5059 AAGGTAGCTT TTCCCAAACT GATCTTTTCA TTTAGGTGAG GAAGCAAAAGCATCTATGTG 5119 AGACCATTTA GCACACTGCT TGTGAAAGGA AAGAGGCTCT GGCTAAATTCATGCTGCTTA 5179 GATGACATCT GTCTAGGAAT CATGTGCCAA GCAGAGGTTG GGAGGCCATTTGTGTTTATA 5239 TATAAGCCAA AAAATGCTTG CTTCAACCCC ATGAGACTCG ATAGTGGTGGTGAACAGAAC 5299 AAAAGGTCAT TGGTGGCAGA GTGGATTCTT GAACAAACTG GAAAGTACGTTATGATAGTG 5359 TCCCACGGTG CCTTGGGGAC AAGAGCAGGT GGATTGTGCG TGCATGTGTGTTCATGCACA 5419 CTTGCACCCA TGTGTAGTCA GGTGCCTCAA GAGAAGGCAA CCTTGACTCTTTCTATTGTT 5479 TCTTTCAATA TCCCCAAGCA GTGTGATTGT TTGGCTTATA TACAGACAGAGATGGCCATG 5539 TATTACCTGA ATTTTGGCTG TGTCTCCCTT CATCCTTCTG GAATAAGGAGAATGAAAATT 5599 CTTGATAAAG AAGATTCTGT GGTCTAAACA AAAAAAGGCG GTGAGCAATCCTGCAAGAGC 5659 AAGGTACATA AACAAGTCCT CAGTGGTTGG CAACTGTTTC AACCTGTTTGAACCAAGAAC 5719 CTTCCAGGAA GGCTAAAGGG AAACCGAATT TCACAGCCAT GATTCTTTTGCCCACACTTG 5779 GGAGCAAAAG ATTCTACAAA GCTCTTTTGA GCATTTAGAC TCTCGACTGGCCAAGGTTTG 5839 GGGAAGAACG AAGCCACCTT TGAAGAAGTA AGGAGTCGTG TATGGTAGGGTAAGTGAGAG 5899 AGGGGGATGT TTCCAATGCT TTGATCCCTT CTTACTTAAC CTGAAGCTAGACGAGCAGGC 5959 TTCTTCCCCC CAAAACTGAA TTCC 5983 1484 amino acids aminoacid linear protein 2 Met Lys Pro Arg Ala Glu Cys Cys Ser Pro Lys PheTrp Leu Val Leu 1 5 10 15 Ala Val Leu Ala Val Ser Gly Ser Arg Ala ArgSer Gln Lys Ser Pro 20 25 30 Pro Ser Ile Gly Ile Ala Val Ile Leu Val GlyThr Ser Asp Glu Val 35 40 45 Ala Ile Lys Asp Ala His Glu Lys Asp Asp PheHis His Leu Ser Val 50 55 60 Val Pro Arg Val Glu Leu Val Ala Met Asn GluThr Asp Pro Lys Ser 65 70 75 80 Ile Ile Thr Arg Ile Cys Asp Leu Met SerAsp Arg Lys Ile Gln Gly 85 90 95 Val Val Phe Ala Asp Asp Thr Asp Gln GluAla Ile Ala Gln Ile Leu 100 105 110 Asp Phe Ile Ser Ala Gln Thr Leu ThrPro Ile Leu Gly Ile His Gly 115 120 125 Gly Ser Ser Met Ile Met Ala AspLys Asp Glu Ser Ser Met Phe Phe 130 135 140 Gln Phe Gly Pro Ser Ile GluGln Gln Ala Ser Val Met Leu Asn Ile 145 150 155 160 Met Glu Glu Tyr AspTrp Tyr Ile Phe Ser Ile Val Thr Thr Tyr Phe 165 170 175 Pro Gly Tyr GlnAsp Phe Val Asn Lys Ile Arg Ser Thr Ile Glu Asn 180 185 190 Ser Phe ValGly Trp Glu Leu Glu Glu Val Leu Leu Leu Asp Met Ser 195 200 205 Leu AspAsp Gly Asp Ser Lys Ile Gln Asn Gln Leu Lys Lys Leu Gln 210 215 220 SerPro Ile Ile Leu Leu Tyr Cys Thr Lys Glu Glu Ala Thr Tyr Ile 225 230 235240 Phe Glu Val Ala Asn Ser Val Gly Leu Thr Gly Tyr Gly Tyr Thr Trp 245250 255 Ile Val Pro Ser Leu Val Ala Gly Asp Thr Asp Thr Val Pro Ala Glu260 265 270 Phe Pro Thr Gly Leu Ile Ser Val Ser Tyr Asp Glu Trp Asp TyrGly 275 280 285 Leu Pro Ala Arg Val Arg Asp Gly Ile Ala Ile Ile Thr ThrAla Ala 290 295 300 Ser Asp Met Leu Ser Glu His Ser Phe Ile Pro Glu ProLys Ser Ser 305 310 315 320 Cys Tyr Asn Thr His Glu Lys Arg Ile Tyr GlnSer Asn Met Leu Asn 325 330 335 Arg Tyr Leu Ile Asn Val Thr Phe Glu GlyArg Asn Leu Ser Phe Ser 340 345 350 Glu Asp Gly Tyr Gln Met His Pro LysLeu Val Ile Ile Leu Leu Asn 355 360 365 Lys Glu Arg Lys Trp Glu Arg ValGly Lys Trp Lys Asp Lys Ser Leu 370 375 380 Gln Met Lys Tyr Tyr Val TrpPro Arg Met Cys Pro Glu Thr Glu Glu 385 390 395 400 Gln Glu Asp Asp HisLeu Ser Ile Val Thr Leu Glu Glu Ala Pro Phe 405 410 415 Val Ile Val GluSer Val Asp Pro Leu Ser Gly Thr Cys Met Arg Asn 420 425 430 Thr Val ProCys Gln Lys Arg Ile Val Thr Glu Asn Lys Thr Asp Glu 435 440 445 Glu ProGly Tyr Ile Lys Lys Cys Cys Lys Gly Phe Cys Ile Asp Ile 450 455 460 LeuLys Lys Ile Ser Lys Ser Val Lys Phe Thr Tyr Asp Leu Tyr Leu 465 470 475480 Val Thr Asn Gly Lys His Gly Lys Lys Ile Asn Gly Thr Trp Asn Gly 485490 495 Met Ile Gly Glu Val Val Met Lys Arg Ala Tyr Met Ala Val Gly Ser500 505 510 Leu Thr Ile Asn Glu Glu Arg Ser Glu Val Val Asp Phe Ser ValPro 515 520 525 Phe Ile Glu Thr Gly Ile Ser Val Met Val Ser Arg Ser AsnGly Thr 530 535 540 Val Ser Pro Ser Ala Phe Leu Glu Pro Phe Ser Ala AspVal Trp Val 545 550 555 560 Met Met Phe Val Met Leu Leu Ile Val Ser AlaVal Ala Val Phe Val 565 570 575 Phe Glu Tyr Phe Ser Pro Val Gly Tyr AsnArg Cys Leu Ala Asp Gly 580 585 590 Arg Glu Pro Gly Gly Pro Ser Phe ThrIle Gly Lys Ala Ile Trp Leu 595 600 605 Leu Trp Gly Leu Val Phe Asn AsnSer Val Pro Val Gln Asn Pro Lys 610 615 620 Gly Thr Thr Ser Lys Ile MetVal Ser Val Trp Ala Phe Phe Ala Val 625 630 635 640 Ile Phe Leu Ala SerTyr Thr Ala Asn Leu Ala Ala Phe Met Ile Gln 645 650 655 Glu Glu Tyr ValAsp Gln Val Ser Gly Leu Ser Asp Lys Lys Phe Gln 660 665 670 Arg Pro AsnAsp Phe Ser Pro Pro Phe Arg Phe Gly Thr Val Pro Asn 675 680 685 Gly SerThr Glu Arg Asn Ile Arg Asn Asn Tyr Ala Glu Met His Ala 690 695 700 TyrMet Gly Lys Phe Asn Gln Arg Gly Val Asp Asp Ala Leu Leu Ser 705 710 715720 Leu Lys Thr Gly Lys Leu Asp Ala Phe Ile Tyr Asp Ala Ala Val Leu 725730 735 Asn Tyr Met Ala Gly Arg Asp Glu Gly Cys Lys Leu Val Thr Ile Gly740 745 750 Ser Gly Lys Val Phe Ala Ser Thr Gly Tyr Gly Ile Ala Ile GlnLys 755 760 765 Asp Ser Gly Trp Lys Arg Gln Val Asp Leu Ala Ile Leu GlnLeu Phe 770 775 780 Gly Asp Gly Glu Met Glu Glu Leu Glu Ala Leu Trp LeuThr Gly Ile 785 790 795 800 Cys His Asn Glu Lys Asn Glu Val Met Ser SerGln Leu Asp Ile Asp 805 810 815 Asn Met Ala Gly Val Phe Tyr Met Leu GlyAla Ala Met Ala Leu Ser 820 825 830 Leu Ile Thr Phe Ile Cys Glu His LeuPhe Tyr Trp Gln Phe Arg His 835 840 845 Cys Phe Met Gly Val Cys Ser GlyLys Pro Gly Met Val Phe Ser Ile 850 855 860 Ser Arg Gly Ile Tyr Ser CysIle His Gly Val Ala Ile Glu Glu Arg 865 870 875 880 Gln Ser Val Met AsnSer Pro Thr Ala Thr Met Asn Asn Thr His Ser 885 890 895 Asn Ile Leu ArgLeu Leu Arg Thr Ala Lys Asn Met Ala Asn Leu Ser 900 905 910 Gly Val AsnGly Ser Pro Gln Arg Pro Leu Asp Phe Ile Arg Arg Glu 915 920 925 Ser SerVal Tyr Asp Ile Ser Glu His Arg Arg Ser Phe Thr His Ser 930 935 940 AspCys Lys Ser Tyr Asn Asn Pro Pro Cys Glu Glu Asn Leu Phe Ser 945 950 955960 Asp Tyr Ile Ser Glu Val Glu Arg Thr Phe Gly Asn Leu Gln Leu Lys 965970 975 Asp Ser Asn Val Tyr Gln Asp His Tyr His His His His Arg Pro His980 985 990 Ser Ile Gly Ser Ala Ser Ser Ile Asp Gly Leu Tyr Asp Cys AspAsn 995 1000 1005 Pro Pro Phe Thr Thr Gln Ser Arg Ser Ile Ser Lys LysPro Leu Asp 1010 1015 1020 Ile Gly Leu Pro Ser Ser Lys His Ser Gln LeuSer Asp Leu Tyr Gly 1025 1030 1035 1040 Lys Phe Ser Phe Lys Ser Asp ArgTyr Ser Gly His Asp Asp Leu Ile 1045 1050 1055 Arg Ser Asp Val Ser AspIle Ser Thr His Thr Val Thr Tyr Gly Asn 1060 1065 1070 Ile Glu Gly AsnAla Ala Lys Arg Arg Lys Gln Gln Tyr Lys Asp Ser 1075 1080 1085 Leu LysLys Arg Pro Ala Ser Ala Lys Ser Arg Arg Glu Phe Asp Glu 1090 1095 1100Ile Glu Leu Ala Tyr Arg Arg Arg Pro Pro Arg Ser Pro Asp His Lys 11051110 1115 1120 Arg Tyr Phe Arg Asp Lys Glu Gly Leu Arg Asp Phe Tyr LeuAsp Gln 1125 1130 1135 Phe Arg Thr Lys Glu Asn Ser Pro His Trp Glu HisVal Asp Leu Thr 1140 1145 1150 Asp Ile Tyr Lys Glu Arg Ser Asp Asp PheLys Arg Asp Ser Val Ser 1155 1160 1165 Gly Gly Gly Pro Cys Thr Asn ArgSer His Ile Lys His Gly Thr Gly 1170 1175 1180 Asp Lys His Gly Val ValSer Gly Val Pro Ala Pro Trp Glu Lys Asn 1185 1190 1195 1200 Leu Thr AsnVal Glu Trp Glu Asp Arg Ser Gly Gly Asn Phe Cys Arg 1205 1210 1215 SerCys Pro Ser Lys Leu His Asn Tyr Ser Thr Thr Val Thr Gly Gln 1220 12251230 Asn Ser Gly Arg Gln Ala Cys Ile Arg Cys Glu Ala Cys Lys Lys Ala1235 1240 1245 Gly Asn Leu Tyr Asp Ile Ser Glu Asp Asn Ser Leu Gln GluLeu Asp 1250 1255 1260 Gln Pro Ala Ala Pro Val Ala Val Thr Ser Asn AlaSer Thr Thr Lys 1265 1270 1275 1280 Tyr Pro Gln Ser Pro Thr Asn Ser LysAla Gln Lys Lys Asn Arg Asn 1285 1290 1295 Lys Leu Arg Arg Gln His SerTyr Asp Thr Phe Val Asp Leu Gln Lys 1300 1305 1310 Glu Glu Ala Ala LeuAla Pro Arg Ser Val Ser Leu Lys Asp Lys Gly 1315 1320 1325 Arg Phe MetAsp Gly Ser Pro Tyr Ala His Met Phe Glu Met Ser Ala 1330 1335 1340 GlyGlu Ser Thr Phe Ala Asn Asn Lys Ser Ser Val Pro Thr Ala Gly 1345 13501355 1360 His His His His Asn Asn Pro Gly Gly Gly Tyr Met Leu Ser LysSer 1365 1370 1375 Leu Tyr Pro Asp Arg Val Thr Gln Asn Pro Phe Ile ProThr Phe Gly 1380 1385 1390 Asp Asp Gln Cys Leu Leu His Gly Ser Lys SerTyr Phe Phe Arg Gln 1395 1400 1405 Pro Thr Val Ala Gly Ala Ser Lys AlaArg Pro Asp Phe Arg Ala Leu 1410 1415 1420 Val Thr Asn Lys Pro Val ValSer Ala Leu His Gly Ala Val Pro Ala 1425 1430 1435 1440 Arg Phe Gln LysAsp Ile Cys Ile Gly Asn Gln Ser Asn Pro Cys Val 1445 1450 1455 Pro AsnAsn Lys Asn Pro Arg Ala Phe Asn Gly Ser Ser Asn Gly His 1460 1465 1470Val Tyr Glu Lys Leu Ser Ser Ile Glu Ser Asp Val 1475 1480 20 amino acidsamino acid linear 3 Tyr Val Trp Pro Arg Met Cys Pro Glu Thr Glu Glu GlnGlu Asp Asp 1 5 10 15 His Leu Ser Ile 20 60 base pairs nucleic aciddouble linear 4 CTATGTGTGG CCCCGAATGT GTCCAGAGAC TGAAGAGCAG GAGGATGACCATCTGAGCAT 60 60 base pairs nucleic acid double linear 5 CTATGTGTGGCCCCGAATGT GTCCAGAGAC TGAAGAGCAG GAGGATGACC ATCTGAACAT 60 20 amino acidsamino acid linear 6 Tyr Val Trp Pro Arg Met Cys Pro Glu Thr Glu Glu GlnGlu Asp Asp 1 5 10 15 His Leu Asn Ile 20 1785 base pairs nucleic aciddouble linear CDS 1..1785 7 GCC TTC TAC AGG CAC CTA CTG AAT GTC ACC TGGGAG GGC CGA GAC TTC 48 Ala Phe Tyr Arg His Leu Leu Asn Val Thr Trp GluGly Arg Asp Phe 1 5 10 15 TCC TTC AGC CCT GGT GGG TAC CTG GTC CAG CCCACC ATG GTG GTG ATC 96 Ser Phe Ser Pro Gly Gly Tyr Leu Val Gln Pro ThrMet Val Val Ile 20 25 30 GCC CTC AAC CGG CAC CGC CTC TGG GAG ATG GTG GGGCGC TGG GAG CAT 144 Ala Leu Asn Arg His Arg Leu Trp Glu Met Val Gly ArgTrp Glu His 35 40 45 GGC GTC CTA TAC ATG AAG TAC CCC GTG TGG CCT CGC TACAGT GCC TCT 192 Gly Val Leu Tyr Met Lys Tyr Pro Val Trp Pro Arg Tyr SerAla Ser 50 55 60 CTG CAG CCT GTG GTG GAC AGT CGG CAC CTG ACG GTG GCC ACGCTG GAA 240 Leu Gln Pro Val Val Asp Ser Arg His Leu Thr Val Ala Thr LeuGlu 65 70 75 80 GAG CGG CCC TTT GTC ATC GTG GAG AGC CCT GAC CCT GGC ACAGGA GGC 288 Glu Arg Pro Phe Val Ile Val Glu Ser Pro Asp Pro Gly Thr GlyGly 85 90 95 TGT GTC CCC AAC ACC GTG CCC TGC CGC AGG CAG AGC AAC CAC ACCTTC 336 Cys Val Pro Asn Thr Val Pro Cys Arg Arg Gln Ser Asn His Thr Phe100 105 110 AGC AGC GGG GAC GTG GCC CCC TAC ACC AAG CTC TGC TGT AAG GGATTC 384 Ser Ser Gly Asp Val Ala Pro Tyr Thr Lys Leu Cys Cys Lys Gly Phe115 120 125 TGC ATC GAC ATC CTC AAG AAG CTG GCC AGA GTG GTC AAA TTC TCCTAC 432 Cys Ile Asp Ile Leu Lys Lys Leu Ala Arg Val Val Lys Phe Ser Tyr130 135 140 GAC CTG TAC CTG GTG ACC AAC GGC AAG CAT GGC AAG CGG GTG CGCGGC 480 Asp Leu Tyr Leu Val Thr Asn Gly Lys His Gly Lys Arg Val Arg Gly145 150 155 160 GTA TGG AAC GGC ATG ATT GGG GAG GTG TAC TAC AAG CGG GCAGAC ATG 528 Val Trp Asn Gly Met Ile Gly Glu Val Tyr Tyr Lys Arg Ala AspMet 165 170 175 GCC ATC GGC TCC CTC ACC ATC AAT GAG GAA CGC TCC GAG ATCGTA GAC 576 Ala Ile Gly Ser Leu Thr Ile Asn Glu Glu Arg Ser Glu Ile ValAsp 180 185 190 TTC TCT GTA CCC TTT GTG GAG ACG GGC ATC AGT GTG ATG GTGGCT CGC 624 Phe Ser Val Pro Phe Val Glu Thr Gly Ile Ser Val Met Val AlaArg 195 200 205 AGC AAT GGC ACC GTC TCC CCC TCG GCC TTC TTG GAG CCA TATAGC CCT 672 Ser Asn Gly Thr Val Ser Pro Ser Ala Phe Leu Glu Pro Tyr SerPro 210 215 220 GCA GTG TGG GTG ATG ATG TTT GTC ATG TGC CTC ACT GTG GTGGCC ATC 720 Ala Val Trp Val Met Met Phe Val Met Cys Leu Thr Val Val AlaIle 225 230 235 240 ACC GTC TTC ATG TTC GAG TAC TTC AGC CCT GTC AGC TACAAC CAG AAC 768 Thr Val Phe Met Phe Glu Tyr Phe Ser Pro Val Ser Tyr AsnGln Asn 245 250 255 CTC ACC AGA GGC AAG AAG TCC GGG GGC CCA GCT TTC ACTATC GGC AAG 816 Leu Thr Arg Gly Lys Lys Ser Gly Gly Pro Ala Phe Thr IleGly Lys 260 265 270 TCC GTG TGG CTG CTG TGG GCG CTG GTC TTC AAC AAC TCAGTG CCC ATC 864 Ser Val Trp Leu Leu Trp Ala Leu Val Phe Asn Asn Ser ValPro Ile 275 280 285 GAG AAC CCG CGG GGC ACC ACC AGC AAG ATC ATG GTT CTGGTC TGG GCC 912 Glu Asn Pro Arg Gly Thr Thr Ser Lys Ile Met Val Leu ValTrp Ala 290 295 300 TTC TTT GCT GTC ATC TTC CTC GCC AGC TAC ACG GCC AACCTG GCC GCC 960 Phe Phe Ala Val Ile Phe Leu Ala Ser Tyr Thr Ala Asn LeuAla Ala 305 310 315 320 TTC ATG ATC CAA GAG CAA TAC ATC GAC ACT GTG TCGGGC CTC AGT GAC 1008 Phe Met Ile Gln Glu Gln Tyr Ile Asp Thr Val Ser GlyLeu Ser Asp 325 330 335 AAG AAG TTT CAG CGG CCT CAA GAT CAG TAC CCA CCTTTC CGC TTC GGC 1056 Lys Lys Phe Gln Arg Pro Gln Asp Gln Tyr Pro Pro PheArg Phe Gly 340 345 350 ACG GTG CCC AAC GGC AGC ACG GAG CGG AAC ATC CGCAGT AAC TAC CGT 1104 Thr Val Pro Asn Gly Ser Thr Glu Arg Asn Ile Arg SerAsn Tyr Arg 355 360 365 GAC ATG CAC ACC CAC ATG GTC AAG TTC AAC CAG CGCTCG GTG GAG GAC 1152 Asp Met His Thr His Met Val Lys Phe Asn Gln Arg SerVal Glu Asp 370 375 380 GCG CTC ACC AGC CTC AAG ATG GGG AAG CTG GAT GCCTTC ATC TAT GAT 1200 Ala Leu Thr Ser Leu Lys Met Gly Lys Leu Asp Ala PheIle Tyr Asp 385 390 395 400 GCT GCT GTC CTC AAC TAC ATG GCA GGC AAG GACGAG GGC TGC AAG CTG 1248 Ala Ala Val Leu Asn Tyr Met Ala Gly Lys Asp GluGly Cys Lys Leu 405 410 415 GTC ACC ATT GGG TCT GGC AAG GTC TTT GCT ACCACT GGC TAC GGC ATC 1296 Val Thr Ile Gly Ser Gly Lys Val Phe Ala Thr ThrGly Tyr Gly Ile 420 425 430 GCC ATG CAG AAG GAC TCC CAC TGG AAG CGG GCCATA GAC CTG GCG CTC 1344 Ala Met Gln Lys Asp Ser His Trp Lys Arg Ala IleAsp Leu Ala Leu 435 440 445 TTG CAG TTC CTG GGG GAC GGA GAG ACA CAG AAACTG GAG ACA GTG TGG 1392 Leu Gln Phe Leu Gly Asp Gly Glu Thr Gln Lys LeuGlu Thr Val Trp 450 455 460 CTC TCA GGG ATC TGC CAG AAT GAG AAG AAC GAGGTG ATG AGC AGC AAG 1440 Leu Ser Gly Ile Cys Gln Asn Glu Lys Asn Glu ValMet Ser Ser Lys 465 470 475 480 CTG GAC ATC GAC AAC ATG GCA GGC GTC TTCTAC ATG CTG CTG GTG GCC 1488 Leu Asp Ile Asp Asn Met Ala Gly Val Phe TyrMet Leu Leu Val Ala 485 490 495 ATG GGG CTG GCC CTG CTG GTC TTC GCC TGGGAG CAC CTG GTC TAC TGG 1536 Met Gly Leu Ala Leu Leu Val Phe Ala Trp GluHis Leu Val Tyr Trp 500 505 510 AAG CTG CGC CAC TCG GTG CCC AAC TCA TCCCAG CTG GAC TTC CTG CTG 1584 Lys Leu Arg His Ser Val Pro Asn Ser Ser GlnLeu Asp Phe Leu Leu 515 520 525 GCT TTC AGC AGG GGC ATC TAC AGC TGC TTCAGC GGG GTG CAG AGC CTC 1632 Ala Phe Ser Arg Gly Ile Tyr Ser Cys Phe SerGly Val Gln Ser Leu 530 535 540 GCC AGC CCA CCG CGG CAG GCC AGC CCG GACCTC ACG GCC AGC TCG GCC 1680 Ala Ser Pro Pro Arg Gln Ala Ser Pro Asp LeuThr Ala Ser Ser Ala 545 550 555 560 CAG GCC AGC GTG CTC AAG ATC GTG CAGGCA GCC CGC GAC ATG GTG ACC 1728 Gln Ala Ser Val Leu Lys Ile Val Gln AlaAla Arg Asp Met Val Thr 565 570 575 ACG GCG GGC GTA AGC AGC TCC CTG GACCGC GCC ACT CGC ACC ATC GAG 1776 Thr Ala Gly Val Ser Ser Ser Leu Asp ArgAla Thr Arg Thr Ile Glu 580 585 590 AAT TGG GGT 1785 Asn Trp Gly 595 595amino acids amino acid linear protein 8 Ala Phe Tyr Arg His Leu Leu AsnVal Thr Trp Glu Gly Arg Asp Phe 1 5 10 15 Ser Phe Ser Pro Gly Gly TyrLeu Val Gln Pro Thr Met Val Val Ile 20 25 30 Ala Leu Asn Arg His Arg LeuTrp Glu Met Val Gly Arg Trp Glu His 35 40 45 Gly Val Leu Tyr Met Lys TyrPro Val Trp Pro Arg Tyr Ser Ala Ser 50 55 60 Leu Gln Pro Val Val Asp SerArg His Leu Thr Val Ala Thr Leu Glu 65 70 75 80 Glu Arg Pro Phe Val IleVal Glu Ser Pro Asp Pro Gly Thr Gly Gly 85 90 95 Cys Val Pro Asn Thr ValPro Cys Arg Arg Gln Ser Asn His Thr Phe 100 105 110 Ser Ser Gly Asp ValAla Pro Tyr Thr Lys Leu Cys Cys Lys Gly Phe 115 120 125 Cys Ile Asp IleLeu Lys Lys Leu Ala Arg Val Val Lys Phe Ser Tyr 130 135 140 Asp Leu TyrLeu Val Thr Asn Gly Lys His Gly Lys Arg Val Arg Gly 145 150 155 160 ValTrp Asn Gly Met Ile Gly Glu Val Tyr Tyr Lys Arg Ala Asp Met 165 170 175Ala Ile Gly Ser Leu Thr Ile Asn Glu Glu Arg Ser Glu Ile Val Asp 180 185190 Phe Ser Val Pro Phe Val Glu Thr Gly Ile Ser Val Met Val Ala Arg 195200 205 Ser Asn Gly Thr Val Ser Pro Ser Ala Phe Leu Glu Pro Tyr Ser Pro210 215 220 Ala Val Trp Val Met Met Phe Val Met Cys Leu Thr Val Val AlaIle 225 230 235 240 Thr Val Phe Met Phe Glu Tyr Phe Ser Pro Val Ser TyrAsn Gln Asn 245 250 255 Leu Thr Arg Gly Lys Lys Ser Gly Gly Pro Ala PheThr Ile Gly Lys 260 265 270 Ser Val Trp Leu Leu Trp Ala Leu Val Phe AsnAsn Ser Val Pro Ile 275 280 285 Glu Asn Pro Arg Gly Thr Thr Ser Lys IleMet Val Leu Val Trp Ala 290 295 300 Phe Phe Ala Val Ile Phe Leu Ala SerTyr Thr Ala Asn Leu Ala Ala 305 310 315 320 Phe Met Ile Gln Glu Gln TyrIle Asp Thr Val Ser Gly Leu Ser Asp 325 330 335 Lys Lys Phe Gln Arg ProGln Asp Gln Tyr Pro Pro Phe Arg Phe Gly 340 345 350 Thr Val Pro Asn GlySer Thr Glu Arg Asn Ile Arg Ser Asn Tyr Arg 355 360 365 Asp Met His ThrHis Met Val Lys Phe Asn Gln Arg Ser Val Glu Asp 370 375 380 Ala Leu ThrSer Leu Lys Met Gly Lys Leu Asp Ala Phe Ile Tyr Asp 385 390 395 400 AlaAla Val Leu Asn Tyr Met Ala Gly Lys Asp Glu Gly Cys Lys Leu 405 410 415Val Thr Ile Gly Ser Gly Lys Val Phe Ala Thr Thr Gly Tyr Gly Ile 420 425430 Ala Met Gln Lys Asp Ser His Trp Lys Arg Ala Ile Asp Leu Ala Leu 435440 445 Leu Gln Phe Leu Gly Asp Gly Glu Thr Gln Lys Leu Glu Thr Val Trp450 455 460 Leu Ser Gly Ile Cys Gln Asn Glu Lys Asn Glu Val Met Ser SerLys 465 470 475 480 Leu Asp Ile Asp Asn Met Ala Gly Val Phe Tyr Met LeuLeu Val Ala 485 490 495 Met Gly Leu Ala Leu Leu Val Phe Ala Trp Glu HisLeu Val Tyr Trp 500 505 510 Lys Leu Arg His Ser Val Pro Asn Ser Ser GlnLeu Asp Phe Leu Leu 515 520 525 Ala Phe Ser Arg Gly Ile Tyr Ser Cys PheSer Gly Val Gln Ser Leu 530 535 540 Ala Ser Pro Pro Arg Gln Ala Ser ProAsp Leu Thr Ala Ser Ser Ala 545 550 555 560 Gln Ala Ser Val Leu Lys IleVal Gln Ala Ala Arg Asp Met Val Thr 565 570 575 Thr Ala Gly Val Ser SerSer Leu Asp Arg Ala Thr Arg Thr Ile Glu 580 585 590 Asn Trp Gly 595 530base pairs nucleic acid single linear 9 TCTGGGTGAT GATGTTTGTG ATGCTGCTCATTGTTTCTGC CATAGCTGTT TTTGTCTTTG 60 AATACTTCAG CCCTGTTGGA TACAACAGAAACTTAGCCAA AGGGAAAGCA CCCCATGGGC 120 CTTCTTTTAC AATTGGAAAA GCTATATGGCTTCTTTGGGG CCTGGTGTTC AATAACTCCG 180 TGCCTGTCCA GAATCCTAAA GGGACCACCAGCAAGATCAT GGTATCTGTA TGGGCCTTCT 240 TCGCTGTCAT ATTCCTGGCT AGCTACACAGCCAATCTGGC TGCCTTCATG ATCCAAGAGG 300 AATTTGTGGA CCAAGTGACC GGCCTCAGTGACAAAAAGTT TCAGAGACCT CATGACTATT 360 CCCCACCTTT TCGATTTGGG ACAGTGCCTAATGGAAGCAC GGAGAGAAAC ATTCGGAATA 420 ACTATCCCTA CATGCATCAG TACATGACCAAATTTAATCA GAAAGGAGTA GAGGACGCCT 480 TGGTCAGCCT GAAAACGGGG AAGCTGGACGCTTTCATCTA CGATGCCGCA 530 4659 base pairs nucleic acid double linear CDS1099..3753 sig_peptide 1099..1152 mat_peptide 1153..3753 misc_feature2781..2838 /function= “transmembrane domain” misc_feature 2895..2958/function= “transmembrane domain” misc_feature 2988..3045 /function=“transmembrane domain” misc_feature 3534..3597 /function= “transmembranedomain” 10 GAATTCCGGT AAGGCTCTGG AAAAGGGGGC GCTGGGAGCG CATTGCGAGGGGGCTGGAGA 60 GGGAGAGAGG AGCGGAAGCT GAGGGTGTGA AACGGCTGGC CCCGAACACACCTCGCGGCG 120 CTCCAGTGAT TCCTGGTGTC CGACCTCAGC CCCAGTCAGT GCGGGTCCAGTTTCCAGGCT 180 CTCGCGGAAG GCCTGGCTGA GCACATGCGG CAGCCACGGT CGCCCTCCCTATTCCTCTTA 240 GCCCGAGGAG GGGGGTCCCA AGTTACATGG CCACGCAGAT GGGGCCTCTCCCTCATTTCT 300 GAACCTTGTG GGGAGGGGAA CCTTGAAGGG AGCGCCCCCC AGAGCCATGGCTTAGGGCCT 360 CCCCCACCCC TCTGGAGCTC CAGTCTGCAA GAGTCAGGAG CCGAAATATCGCTGACTGTG 420 GGTGACGACT CTTGCGCGCA CACACACATA CAAGCGGGCA CGACGCGTTCGGTCCTATTA 480 AAAGGCACGC AAGGGTGCGG CTGCACGCGG TGACACGGAC CCCTCTAACGTTTCCAAACT 540 GAGCTCCCTG CAGGTCCCCG ACAGCACAGG CCCCTGTCCC AGGACCCCTCCAGGCACGCG 600 CTCACACGCA CACGCGCGCT CCCCGGCTCA CGCGCGCTCC GACACACACGCTCACGCGAA 660 CGCAGGCGCA CGCTCTGGCG CGGGAGGCGC CCCTTCGCCT CCGTGTTGGGAAGCGGGGGC 720 GGCGGGAGGG GCAGGAGACG TTGGCCCCGC TCGCGTTTCT GCAGCTGCTGCAGTCGCCGC 780 AGCGTCCGGA CCGGAACCAG CGCCGTCCGC GGAGCCGCCG CCGCCGCCGCCGGGCCCTTT 840 CCAAGCCGGG CGCTCGGAGC TGTGCCCGGC CCCGCTTCAG CACCGCGGACAGCTCCGGCC 900 GCGTGGGGCT GAGCCGAGCC CCCGCGCACG CTTCAGCCCC CTTCCCTCGGCCGACGTCCC 960 GGGACCGCCG CTCCGGGGGA GACGTGGCGT CCGCAGCCCG CGGGGCCGGGCGAGCGCAGG 1020 ACGGCCCGGA AGCCCCGCGG GGGATGCGCC GAGGGCCCGC GTTCGCGCCGCGCAGAGCCA 1080 GGCCCGCGGC CCGAGCCC ATG AGC ACC ATG CGC CTG CTG ACG CTCGCC CTG 1131 Met Ser Thr Met Arg Leu Leu Thr Leu Ala Leu -18 -15 -10 CTGTTC TCC TGC TCC GTC GCC CGT GCC GCG TGC GAC CCC AAG ATC GTC 1179 Leu PheSer Cys Ser Val Ala Arg Ala Ala Cys Asp Pro Lys Ile Val -5 1 5 AAC ATTGGC GCG GTG CTG AGC ACG CGG AAG CAC GAG CAG ATG TTC CGC 1227 Asn Ile GlyAla Val Leu Ser Thr Arg Lys His Glu Gln Met Phe Arg 10 15 20 25 GAG GCCGTG AAC CAG GCC AAC AAG CGG CAC GGC TCC TGG AAG ATT CAG 1275 Glu Ala ValAsn Gln Ala Asn Lys Arg His Gly Ser Trp Lys Ile Gln 30 35 40 CTC AAT GCCACC TCC GTC ACG CAC AAG CCC AAC GCC ATC CAG ATG GCT 1323 Leu Asn Ala ThrSer Val Thr His Lys Pro Asn Ala Ile Gln Met Ala 45 50 55 CTG TCG GTG TGCGAG GAC CTC ATC TCC AGC CAG GTC TAC GCC ATC CTA 1371 Leu Ser Val Cys GluAsp Leu Ile Ser Ser Gln Val Tyr Ala Ile Leu 60 65 70 GTT AGC CAT CCA CCTACC CCC AAC GAC CAC TTC ACT CCC ACC CCT GTC 1419 Val Ser His Pro Pro ThrPro Asn Asp His Phe Thr Pro Thr Pro Val 75 80 85 TCC TAC ACA GCC GGC TTCTAC CGC ATA CCC GTG CTG GGG CTG ACC ACC 1467 Ser Tyr Thr Ala Gly Phe TyrArg Ile Pro Val Leu Gly Leu Thr Thr 90 95 100 105 CGC ATG TCC ATC TACTCG GAC AAG AGC ATC CAC CTG AGC TTC CTG CGC 1515 Arg Met Ser Ile Tyr SerAsp Lys Ser Ile His Leu Ser Phe Leu Arg 110 115 120 ACC GTG CCG CCC TACTCC CAC CAG TCC AGC GTG TGG TTT GAG ATG ATG 1563 Thr Val Pro Pro Tyr SerHis Gln Ser Ser Val Trp Phe Glu Met Met 125 130 135 CGT GTC TAC AGC TGGAAC CAC ATC ATC CTG CTG GTC AGC GAC GAC CAC 1611 Arg Val Tyr Ser Trp AsnHis Ile Ile Leu Leu Val Ser Asp Asp His 140 145 150 GAG GGC CGG GCG GCTCAG AAA CGC CTG GAG ACG CTG CTG GAG GAG CGT 1659 Glu Gly Arg Ala Ala GlnLys Arg Leu Glu Thr Leu Leu Glu Glu Arg 155 160 165 GAG TCC AAG GCA GAGAAG GTG CTG CAG TTT GAC CCA GGG ACC AAG AAC 1707 Glu Ser Lys Ala Glu LysVal Leu Gln Phe Asp Pro Gly Thr Lys Asn 170 175 180 185 GTG ACG GCC CTGCTG ATG GAG GCG AAA GAG CTG GAG GCC CGG GTC ATC 1755 Val Thr Ala Leu LeuMet Glu Ala Lys Glu Leu Glu Ala Arg Val Ile 190 195 200 ATC CTT TCT GCCAGC GAG GAC GAT GCT GCC ACT GTA TAC CGC GCA GCC 1803 Ile Leu Ser Ala SerGlu Asp Asp Ala Ala Thr Val Tyr Arg Ala Ala 205 210 215 GCG ATG CTG AACATG ACG GGC TCC GGG TAC GTG TGG CTG GTC GGC GAG 1851 Ala Met Leu Asn MetThr Gly Ser Gly Tyr Val Trp Leu Val Gly Glu 220 225 230 CGC GAG ATC TCGGGG AAC GCC CTG CGC TAC GCC CCA GAC GGC ATC CTC 1899 Arg Glu Ile Ser GlyAsn Ala Leu Arg Tyr Ala Pro Asp Gly Ile Leu 235 240 245 GGG CTG CAG CTCATC AAC GGC AAG AAC GAG TCG GCC CAC ATC AGC GAC 1947 Gly Leu Gln Leu IleAsn Gly Lys Asn Glu Ser Ala His Ile Ser Asp 250 255 260 265 GCC GTG GGCGTG GTG GCC CAG GCC GTG CAC GAG CTC CTC GAG AAG GAG 1995 Ala Val Gly ValVal Ala Gln Ala Val His Glu Leu Leu Glu Lys Glu 270 275 280 AAC ATC ACCGAC CCG CCG CGG GGC TGC GTG GGC AAC ACC AAC ATC TGG 2043 Asn Ile Thr AspPro Pro Arg Gly Cys Val Gly Asn Thr Asn Ile Trp 285 290 295 AAG ACC GGGCCG CTC TTC AAG AGA GTG CTG ATG TCT TCC AAG TAT GCG 2091 Lys Thr Gly ProLeu Phe Lys Arg Val Leu Met Ser Ser Lys Tyr Ala 300 305 310 GAT GGG GTGACT GGT CGC GTG GAG TTC AAT GAG GAT GGG GAC CGG AAG 2139 Asp Gly Val ThrGly Arg Val Glu Phe Asn Glu Asp Gly Asp Arg Lys 315 320 325 TTC GCC AACTAC AGC ATC ATG AAC CTG CAG AAC CGC AAG CTG GTG CAA 2187 Phe Ala Asn TyrSer Ile Met Asn Leu Gln Asn Arg Lys Leu Val Gln 330 335 340 345 GTG GGCATC TAC AAT GGC ACC CAC GTC ATC CCT AAT GAC AGG AAG ATC 2235 Val Gly IleTyr Asn Gly Thr His Val Ile Pro Asn Asp Arg Lys Ile 350 355 360 ATC TGGCCA GGC GGA GAG ACA GAG AAG CCT CGA GGG TAC CAG ATG TCC 2283 Ile Trp ProGly Gly Glu Thr Glu Lys Pro Arg Gly Tyr Gln Met Ser 365 370 375 ACC AGACTG AAG ATT GTG ACG ATC CAC CAG GAG CCC TTC GTG TAC GTC 2331 Thr Arg LeuLys Ile Val Thr Ile His Gln Glu Pro Phe Val Tyr Val 380 385 390 AAG CCCACG CTG AGT GAT GGG ACA TGC AAG GAG GAG TTC ACA GTC AAC 2379 Lys Pro ThrLeu Ser Asp Gly Thr Cys Lys Glu Glu Phe Thr Val Asn 395 400 405 GGC GACCCA GTC AAG AAG GTG ATC TGC ACC GGG CCC AAC GAC ACG TCG 2427 Gly Asp ProVal Lys Lys Val Ile Cys Thr Gly Pro Asn Asp Thr Ser 410 415 420 425 CCGGGC AGC CCC CGC CAC ACG GTG CCT CAG TGT TGC TAC GGC TTT TGC 2475 Pro GlySer Pro Arg His Thr Val Pro Gln Cys Cys Tyr Gly Phe Cys 430 435 440 ATCGAC CTG CTC ATC AAG CTG GCA CGG ACC ATG AAC TTC ACC TAC GAG 2523 Ile AspLeu Leu Ile Lys Leu Ala Arg Thr Met Asn Phe Thr Tyr Glu 445 450 455 GTGCAC CTG GTG GCA GAT GGC AAG TTC GGC ACA CAG GAG CGG GTG AAC 2571 Val HisLeu Val Ala Asp Gly Lys Phe Gly Thr Gln Glu Arg Val Asn 460 465 470 AACAGC AAC AAG AAG GAG TGG AAT GGG ATG ATG GGC GAG CTG CTC AGC 2619 Asn SerAsn Lys Lys Glu Trp Asn Gly Met Met Gly Glu Leu Leu Ser 475 480 485 GGGCAG GCA GAC ATG ATC GTG GCG CCG CTA ACC ATA AAC AAC GAG CGC 2667 Gly GlnAla Asp Met Ile Val Ala Pro Leu Thr Ile Asn Asn Glu Arg 490 495 500 505GCG CAG TAC ATC GAG TTT TCC AAG CCC TTC AAG TAC CAG GGC CTG ACT 2715 AlaGln Tyr Ile Glu Phe Ser Lys Pro Phe Lys Tyr Gln Gly Leu Thr 510 515 520ATT CTG GTC AAG AAG GAG ATT CCC CGG AGC ACG CTG GAC TCG TTC ATG 2763 IleLeu Val Lys Lys Glu Ile Pro Arg Ser Thr Leu Asp Ser Phe Met 525 530 535CAG CCG TTC CAG AGC ACA CTG TGG CTG CTG GTG GGG CTG TCG GTG CAC 2811 GlnPro Phe Gln Ser Thr Leu Trp Leu Leu Val Gly Leu Ser Val His 540 545 550GTG GTG GCC GTG ATG CTG TAC CTG CTG GAC CGC TTC AGC CCC TTC GGC 2859 ValVal Ala Val Met Leu Tyr Leu Leu Asp Arg Phe Ser Pro Phe Gly 555 560 565CGG TTC AAG GTG AAC AGC GAG GAG GAG GAG GAG GAC GCA CTG ACC CTG 2907 ArgPhe Lys Val Asn Ser Glu Glu Glu Glu Glu Asp Ala Leu Thr Leu 570 575 580585 TCC TCG GCC ATG TGG TTC TCC TGG GGC GTC CTG CTC AAC TCC GGC ATC 2955Ser Ser Ala Met Trp Phe Ser Trp Gly Val Leu Leu Asn Ser Gly Ile 590 595600 GGG GAA GGC GCC CCC AGA AGC TTC TCA GCG CGC ATC CTG GGC ATG GTG 3003Gly Glu Gly Ala Pro Arg Ser Phe Ser Ala Arg Ile Leu Gly Met Val 605 610615 TGG GCC GGC TTT GCC ATG ATC ATC GTG GCC TCC TAC ACC GCC AAC CTG 3051Trp Ala Gly Phe Ala Met Ile Ile Val Ala Ser Tyr Thr Ala Asn Leu 620 625630 GCG GCC TTC CTG GTG CTG GAC CGG CCG GAG GAG CGC ATC ACG GGC ATC 3099Ala Ala Phe Leu Val Leu Asp Arg Pro Glu Glu Arg Ile Thr Gly Ile 635 640645 AAC GAC CCT CGG CTG AGG AAC CCC TCG GAC AAG TTT ATC TAC GCC ACG 3147Asn Asp Pro Arg Leu Arg Asn Pro Ser Asp Lys Phe Ile Tyr Ala Thr 650 655660 665 GTG AAG CAG AGC TCC GTG GAT ATC TAC TTC CGG CGC CAG GTG GAG CTG3195 Val Lys Gln Ser Ser Val Asp Ile Tyr Phe Arg Arg Gln Val Glu Leu 670675 680 AGC ACC ATG TAC CGG CAT ATG GAG AAG CAC AAC TAC GAG AGT GCG GCG3243 Ser Thr Met Tyr Arg His Met Glu Lys His Asn Tyr Glu Ser Ala Ala 685690 695 GAG GCC ATC CAG GCC GTG AGA GAC AAC AAG CTG CAT GCC TTC ATC TGG3291 Glu Ala Ile Gln Ala Val Arg Asp Asn Lys Leu His Ala Phe Ile Trp 700705 710 GAC TCG GCG GTG CTG GAG TTC GAG GCC TCG CAG AAG TGC GAC CTG GTG3339 Asp Ser Ala Val Leu Glu Phe Glu Ala Ser Gln Lys Cys Asp Leu Val 715720 725 ACG ACT GGA GAG CTG TTT TTC CGC TCG GGC TTC GGC ATA GGC ATG CGC3387 Thr Thr Gly Glu Leu Phe Phe Arg Ser Gly Phe Gly Ile Gly Met Arg 730735 740 745 AAA GAC AGC CCC TGG AAG CAG AAC GTC TCC CTG TCC ATC CTC AAGTCC 3435 Lys Asp Ser Pro Trp Lys Gln Asn Val Ser Leu Ser Ile Leu Lys Ser750 755 760 CAC GAG AAT GGC TTC ATG GAA GAC CTG GAC AAG ACG TGG GTT CGGTAT 3483 His Glu Asn Gly Phe Met Glu Asp Leu Asp Lys Thr Trp Val Arg Tyr765 770 775 CAG GAA TGT GAC TCG CGC AGC AAC GCC CCT GCG ACC CTT ACT TTTGAG 3531 Gln Glu Cys Asp Ser Arg Ser Asn Ala Pro Ala Thr Leu Thr Phe Glu780 785 790 AAC ATG GCC GGG GTC TTC ATG CTG GTA GCT GGG GGC ATC GTG GCCGGG 3579 Asn Met Ala Gly Val Phe Met Leu Val Ala Gly Gly Ile Val Ala Gly795 800 805 ATC TTC CTG ATT TTC ATC GAG ATT GCC TAC AAG CGG CAC AAG GATGCT 3627 Ile Phe Leu Ile Phe Ile Glu Ile Ala Tyr Lys Arg His Lys Asp Ala810 815 820 825 CGC CGG AAG CAG ATG CAG CTG GCC TTT GCC GCC GTT AAC GTGTGG CGG 3675 Arg Arg Lys Gln Met Gln Leu Ala Phe Ala Ala Val Asn Val TrpArg 830 835 840 AAG AAC CTG CAG CAG TAC CAT CCC ACT GAT ATC ACG GGC CCGCTC AAC 3723 Lys Asn Leu Gln Gln Tyr His Pro Thr Asp Ile Thr Gly Pro LeuAsn 845 850 855 CTC TCA GAT CCC TCG GTC AGC ACC GTG GTG TGAGGCCCCCGGAGGCGCCC 3773 Leu Ser Asp Pro Ser Val Ser Thr Val Val 860 865ACCTGCCCAG TTAGCCCGGC CAAGGACACT GATGGGTCCT GCTGCTCGGG AAGGCCTGAG 3833GGAAGCCCAC CCGCCCCAGA GACTGCCCAC CCTGGGCCTC CCGTCCGTCC GCCCGCCCAC 3893CCCGCTGCCT GGCGGGCAGC CCCTGCTGGA CCAAGGTGCG GACCGGAGCG GCTGAGGACG 3953GGGCAGAGCT GAGTCGGCTG GGCAGGGCGC AGGGCGCTCC GGCAGAGGCA GGGCCCTGGG 4013GTCTCTGAGC AGTGGGGAGC GGGGGCTAAC TGGCCCCAGG CGAAGGGGCT TGGAGCAGAG 4073ACGGCAGCCC CATCCTTCCC GCAGCACCAG CCTGAGCCAC AGTGGGGCCC ATGGCCCCAG 4133CTGGCTGGGT CGCCCCTCCT CGGGCGCCTG CGCTCCTCTG CAGCCTGAGC TCCACCCTCC 4193CCTCTTCTTG CGGCACCGCC CACCCACACC CCGTCTGCCC CTTGACCCCA CACGCCGGGG 4253CTGGCCCTGC CCTCCCCCAC GGCCGTCCCT GACTTCCCAG CTGGCAGCGC CTCCCGCCGC 4313CTCGGGCCGC CTCCTCCAGA CTCGAGAGGG CTGAGCCCCT CCTCTCCTCG TCCGGCCTGC 4373AGCCCAGAAC GGGCCTCCCC GGGGGTCCCC GGACGCTGGC TCGGGACTGT CTTCAACCCT 4433GCCCTGCACC TTGGGCACGG GAGAGCGCCA CCCGCCCGCC CCCGCCCTCG CTCCGGGTGC 4493GTGACCGGCC CGCCACCTTG TACAGAACCA GCACTCCCAG GGCCCGAGCG CGTGCCTTCC 4553CCGTGCGGCC CGTGCGCAGC CGCGCTCTGC CCCTCCGTCC CCAGGGTGCA GGCGCGCACC 4613GCCCAACCCC CACCTCCCGG TGTATGCAGT GGTGATGCCG GAATTC 4659 885 amino acidsamino acid linear protein 11 Met Ser Thr Met Arg Leu Leu Thr Leu Ala LeuLeu Phe Ser Cys Ser -18 -15 -10 -5 Val Ala Arg Ala Ala Cys Asp Pro LysIle Val Asn Ile Gly Ala Val 1 5 10 Leu Ser Thr Arg Lys His Glu Gln MetPhe Arg Glu Ala Val Asn Gln 15 20 25 30 Ala Asn Lys Arg His Gly Ser TrpLys Ile Gln Leu Asn Ala Thr Ser 35 40 45 Val Thr His Lys Pro Asn Ala IleGln Met Ala Leu Ser Val Cys Glu 50 55 60 Asp Leu Ile Ser Ser Gln Val TyrAla Ile Leu Val Ser His Pro Pro 65 70 75 Thr Pro Asn Asp His Phe Thr ProThr Pro Val Ser Tyr Thr Ala Gly 80 85 90 Phe Tyr Arg Ile Pro Val Leu GlyLeu Thr Thr Arg Met Ser Ile Tyr 95 100 105 110 Ser Asp Lys Ser Ile HisLeu Ser Phe Leu Arg Thr Val Pro Pro Tyr 115 120 125 Ser His Gln Ser SerVal Trp Phe Glu Met Met Arg Val Tyr Ser Trp 130 135 140 Asn His Ile IleLeu Leu Val Ser Asp Asp His Glu Gly Arg Ala Ala 145 150 155 Gln Lys ArgLeu Glu Thr Leu Leu Glu Glu Arg Glu Ser Lys Ala Glu 160 165 170 Lys ValLeu Gln Phe Asp Pro Gly Thr Lys Asn Val Thr Ala Leu Leu 175 180 185 190Met Glu Ala Lys Glu Leu Glu Ala Arg Val Ile Ile Leu Ser Ala Ser 195 200205 Glu Asp Asp Ala Ala Thr Val Tyr Arg Ala Ala Ala Met Leu Asn Met 210215 220 Thr Gly Ser Gly Tyr Val Trp Leu Val Gly Glu Arg Glu Ile Ser Gly225 230 235 Asn Ala Leu Arg Tyr Ala Pro Asp Gly Ile Leu Gly Leu Gln LeuIle 240 245 250 Asn Gly Lys Asn Glu Ser Ala His Ile Ser Asp Ala Val GlyVal Val 255 260 265 270 Ala Gln Ala Val His Glu Leu Leu Glu Lys Glu AsnIle Thr Asp Pro 275 280 285 Pro Arg Gly Cys Val Gly Asn Thr Asn Ile TrpLys Thr Gly Pro Leu 290 295 300 Phe Lys Arg Val Leu Met Ser Ser Lys TyrAla Asp Gly Val Thr Gly 305 310 315 Arg Val Glu Phe Asn Glu Asp Gly AspArg Lys Phe Ala Asn Tyr Ser 320 325 330 Ile Met Asn Leu Gln Asn Arg LysLeu Val Gln Val Gly Ile Tyr Asn 335 340 345 350 Gly Thr His Val Ile ProAsn Asp Arg Lys Ile Ile Trp Pro Gly Gly 355 360 365 Glu Thr Glu Lys ProArg Gly Tyr Gln Met Ser Thr Arg Leu Lys Ile 370 375 380 Val Thr Ile HisGln Glu Pro Phe Val Tyr Val Lys Pro Thr Leu Ser 385 390 395 Asp Gly ThrCys Lys Glu Glu Phe Thr Val Asn Gly Asp Pro Val Lys 400 405 410 Lys ValIle Cys Thr Gly Pro Asn Asp Thr Ser Pro Gly Ser Pro Arg 415 420 425 430His Thr Val Pro Gln Cys Cys Tyr Gly Phe Cys Ile Asp Leu Leu Ile 435 440445 Lys Leu Ala Arg Thr Met Asn Phe Thr Tyr Glu Val His Leu Val Ala 450455 460 Asp Gly Lys Phe Gly Thr Gln Glu Arg Val Asn Asn Ser Asn Lys Lys465 470 475 Glu Trp Asn Gly Met Met Gly Glu Leu Leu Ser Gly Gln Ala AspMet 480 485 490 Ile Val Ala Pro Leu Thr Ile Asn Asn Glu Arg Ala Gln TyrIle Glu 495 500 505 510 Phe Ser Lys Pro Phe Lys Tyr Gln Gly Leu Thr IleLeu Val Lys Lys 515 520 525 Glu Ile Pro Arg Ser Thr Leu Asp Ser Phe MetGln Pro Phe Gln Ser 530 535 540 Thr Leu Trp Leu Leu Val Gly Leu Ser ValHis Val Val Ala Val Met 545 550 555 Leu Tyr Leu Leu Asp Arg Phe Ser ProPhe Gly Arg Phe Lys Val Asn 560 565 570 Ser Glu Glu Glu Glu Glu Asp AlaLeu Thr Leu Ser Ser Ala Met Trp 575 580 585 590 Phe Ser Trp Gly Val LeuLeu Asn Ser Gly Ile Gly Glu Gly Ala Pro 595 600 605 Arg Ser Phe Ser AlaArg Ile Leu Gly Met Val Trp Ala Gly Phe Ala 610 615 620 Met Ile Ile ValAla Ser Tyr Thr Ala Asn Leu Ala Ala Phe Leu Val 625 630 635 Leu Asp ArgPro Glu Glu Arg Ile Thr Gly Ile Asn Asp Pro Arg Leu 640 645 650 Arg AsnPro Ser Asp Lys Phe Ile Tyr Ala Thr Val Lys Gln Ser Ser 655 660 665 670Val Asp Ile Tyr Phe Arg Arg Gln Val Glu Leu Ser Thr Met Tyr Arg 675 680685 His Met Glu Lys His Asn Tyr Glu Ser Ala Ala Glu Ala Ile Gln Ala 690695 700 Val Arg Asp Asn Lys Leu His Ala Phe Ile Trp Asp Ser Ala Val Leu705 710 715 Glu Phe Glu Ala Ser Gln Lys Cys Asp Leu Val Thr Thr Gly GluLeu 720 725 730 Phe Phe Arg Ser Gly Phe Gly Ile Gly Met Arg Lys Asp SerPro Trp 735 740 745 750 Lys Gln Asn Val Ser Leu Ser Ile Leu Lys Ser HisGlu Asn Gly Phe 755 760 765 Met Glu Asp Leu Asp Lys Thr Trp Val Arg TyrGln Glu Cys Asp Ser 770 775 780 Arg Ser Asn Ala Pro Ala Thr Leu Thr PheGlu Asn Met Ala Gly Val 785 790 795 Phe Met Leu Val Ala Gly Gly Ile ValAla Gly Ile Phe Leu Ile Phe 800 805 810 Ile Glu Ile Ala Tyr Lys Arg HisLys Asp Ala Arg Arg Lys Gln Met 815 820 825 830 Gln Leu Ala Phe Ala AlaVal Asn Val Trp Arg Lys Asn Leu Gln Gln 835 840 845 Tyr His Pro Thr AspIle Thr Gly Pro Leu Asn Leu Ser Asp Pro Ser 850 855 860 Val Ser Thr ValVal 865 28 base pairs nucleic acid double linear 12 GAAGAACCTGCAGCAGTACC ATCCCACT 28 391 base pairs nucleic acid double linear 13GAAGAACCTG CAGAGCACCG GGGGTGGACG CGGCGCTTTG CAAAACCAAA AAGACACAGT 60GCTGCCGCGA CGCGCTATTG AGAGGGAGGA GGGCCAGCTG CAGCTGTGTT CCCGTCATAG 120GGAGAGCTGA GACTCCCCGC CCGCCCTCCT CTGCCCCCTC CCCCGCAGAC AGACAGACAG 180ACGGATGGGA CAGCGGCCCG GCCCACGCAG AGCCCCGGAG CACCACGGGG TCGGGGGAGG 240AGCACCCCCA GCCTCCCCCA GGCTGCGCCT GCCCGCCCGC CGGTTGGCCG GCTGGCCGGT 300CCACCCCGTC CCGGCCCCGC GCGTGCCCCC AGCGTGGGGC TAACGGGCGC CTTGTCTGTG 360TATTTCTATT TTGCAGCAGT ACCATCCCAC T 391 502 base pairs nucleic aciddouble linear 14 GAAGAACCTG CAGGATAGAA AGAGTGGTAG AGCAGAGCCT GACCCTAAAAAGAAAGCCAC 60 ATTTAGGGCT ATCACCTCCA CCCTGGCTTC CAGCTTCAAG AGGCGTAGGTCCTCCAAAGA 120 CACGAGCACC GGGGGTGGAC GCGGCGCTTT GCAAAACCAA AAAGACACAGTGCTGCCGCG 180 ACGCGCTATT GAGAGGGAGG AGGGCCAGCT GCAGCTGTGT TCCCGTCATAGGGAGAGCTG 240 AGACTCCCCG CCCGCCCTCC TCTGCCCCCT CCCCCGCAGA CAGACAGACAGACGGATGGG 300 ACAGCGGCCC GGCCCACGCA GAGCCCCGGA GCACCACGGG GTCGGGGGAGGAGCACCCCC 360 AGCCTCCCCC AGGCTGCGCC TGCCCGCCCG CCGGTTGGCC GGCTGGCCGGTCCACCCCGT 420 CCCGGCCCCG CGCGTGCCCC CAGCGTGGGG CTAACGGGCG CCTTGTCTGTGTATTTCTAT 480 TTTGCAGCAG TACCATCCCA CT 502 1004 base pairs nucleic aciddouble linear 15 GAAGAACCTG CAGGATAGAA AGAGTGGTAG AGCAGAGCCT GACCCTAAAAAGAAAGCCAC 60 ATTTAGGGCT ATCACCTCCA CCCTGGCTTC CAGCTTCAAG AGGCGTAGGTCCTCCAAAGA 120 CACGAGCACC GGGGGTGGAC GCGGCGCTTT GCAAAACCAA AAAGACACAGTGCTGCCGCG 180 ACGCGCTATT GAGAGGGAGG AGGGCCAGCT GCAGCTGTGT TCCCGTCATACGGAGAGCTG 240 AGACTCCCCG CCCGCCCTCC TCTGCCCCCT CCCCCGCAGA CAGACAGACAGACGGATGGG 300 ACAGCGGCCC GGCCCACGCA GAGCCCCGGA GCACCACGGG GTCGGGGGAGGAGCACCCCC 360 AGCCTCCCCC AGGCTGCGCC TGCCCGCCCG CCGGTTGGCC GGCTGGCCGGTCCACCCCGT 420 CCCGGCCCCG CGCGTGCCCC CAGCGTGGGG CTAACGGGCG CCTTGTCTGTGTATTTCTAT 480 TTTGCAGCAG TACCATCCCA CTGAAGAACC TGCAGGATAG AAAGAGTGGTAGAGCAGAGC 540 CTGACCCTAA AAAGAAAGCC ACATTTAGGG CTATCACCTC CACCCTGGCTTCCAGCTTCA 600 AGAGGCGTAG GTCCTCCAAA GACACGAGCA CCGGGGGTGG ACGCGGCGCTTTGCAAAACC 660 AAAAAGACAC AGTGCTGCCG CGACGCGCTA TTGAGAGGGA GGAGGGCCAGCTGCAGCTGT 720 GTTCCCGTCA TAGGGAGAGC TGAGACTCCC CGCCCGCCCT CCTCTGCCCCCTCCCCCGCA 780 GACAGACAGA CAGACGGATG GGACAGCGGC CCGGCCCACG CAGAGCCCCGGAGCACCACG 840 GGGTCGGGGG AGGAGCACCC CCAGCCTCCC CCAGGCTGCG CCTGCCCGCCCGCCGGTTGG 900 CCGGCTGGCC GGTCCACCCC GTCCCGGCCC CGCGCGTGCC CCCAGCGTGGGGCTAACGGG 960 CGCCTTGTCT GTGTATTTCT ATTTTGCAGC AGTACCATCC CACT 1004 65amino acids amino acid linear 16 Ala Gly Gly Ile Val Ala Gly Ile Phe LeuIle Phe Ile Glu Ile Ala 1 5 10 15 Tyr Lys Arg His Lys Asp Ala Arg ArgLys Gln Met Gln Leu Ala Phe 20 25 30 Ala Ala Val Asn Val Trp Arg Lys AsnLeu Gln Gln Tyr His Pro Thr 35 40 45 Asp Ile Thr Gly Pro Leu Asn Leu SerAsp Pro Ser Val Ser Thr Val 50 55 60 Val 65 102 amino acids amino acidlinear 17 Ala Gly Gly Ile Val Ala Gly Ile Phe Leu Ile Phe Ile Glu IleAla 1 5 10 15 Tyr Lys Arg His Lys Asp Ala Arg Arg Lys Gln Met Gln LeuAla Phe 20 25 30 Ala Ala Val Asn Val Trp Arg Lys Asn Leu Gln Asp Arg LysSer Gly 35 40 45 Arg Ala Glu Pro Asp Pro Lys Lys Lys Ala Thr Phe Arg AlaIle Thr 50 55 60 Ser Thr Leu Ala Ser Ser Phe Lys Arg Arg Arg Ser Ser LysAsp Thr 65 70 75 80 Gln Tyr His Pro Thr Asp Ile Thr Gly Pro Leu Asn LeuSer Asp Pro 85 90 95 Ser Val Ser Thr Val Val 100 26 amino acids aminoacid linear 18 Lys Arg Leu Glu Thr Leu Leu Glu Glu Arg Glu Ser Lys AlaGlu Lys 1 5 10 15 Val Leu Gln Phe Asp Pro Gly Thr Lys Asn 20 25 47 aminoacids amino acid linear 19 Lys Arg Leu Glu Thr Leu Leu Glu Glu Arg GluSer Lys Ser Lys Lys 1 5 10 15 Arg Asn Tyr Glu Asn Leu Asp Gln Leu SerTyr Asp Asn Lys Arg Gly 20 25 30 Pro Lys Ala Glu Lys Val Leu Gln Phe AspPro Gly Thr Lys Asn 35 40 45 33 base pairs nucleic acid single linear 20GGGGTTTAGA TCTGGGTNAT GATGTTYGTN ATG 33 33 base pairs nucleic acidsingle linear 21 GGGGTTTAGA TCTGCNGCRT CRTADATRAA NGC 33 34 base pairsnucleic acid single linear 22 GGGGTTTGGA TCCAARGART GGAAYGGNAT GATG 3434 base pairs nucleic acid single linear 23 GGGGTTTAAG CTTYTCRTARTTRTGYTTYT CCAT 34

We claim:
 1. An isolated polynucleotide comprising a region that encodesa human NR3-1 modulatory protein, or a functional fragment thereof whichretains the modulatory activity of the NR3-1 protein.
 2. An isolatedpolynucleotide comprising a region encoding a functional variant of ahuman NR3-1 receptor, wherein said variant shares greater than 98.5%amino acid identity with said NR3-1 protein and retains the modulatoryactivity of said NR3-1 protein.
 3. An isolated polynucleotide as definedin claim 2, comprising a region that encodes a human NR3-2 protein.
 4. Arecombinant DNA construct having incorporated therein a polynucleotideas defined in claim
 1. 5. A recombinant DNA construct havingincorporated therein a polynucleotide as defined in claim
 2. 6. A cellthat has been engineered genetically to produce a human NR3 protein or afragent thereof, said cell having incorporated expressibly therein aheterologous polynuclotide as defined in claim
 1. 7. A cell that hasbeen engineered genetically to produce a human NR3 protein or a fragmentthereof, said cell having incorporated expressibly therein aheterologous polynucleotide as defined in claim
 2. 8. A membranepreparation derived from a cell as defined in claim
 6. 9. A membranepreparation derived from a cell as defined in claim
 7. 10. A cell thathas been engineered genetically to produce a heteromeric human receptorcomplex comprising an NR3 protein and an NMDA receptor, said cell havingincorporated expressibly therein a heterologous polynucleotide encodinga human NR3 protein and a heterologous polynucleotide encoding a humanNMDA receptor.
 11. A cell as defined in claim 10 wherein said human NR3protein is selected from the group consisting of the NR3-1 and the NR3-2proteins, and the human NMDA receptor is selected from the groupconsisting of the NMDAR1-1, NMDAR1-2, NMDAR1-3, NMDAR1-4, NMDAR1-5,NMDAR1-6, NMDAR1-7 and NMDAR1-8 receptors.
 12. A process for obtaining asubstantially homogeneous source of a human NR3 protein selected fromthe group consisting of the NR3-1 and NR3-2 proteins, said processcomprising the steps of culturing cells having incorporated expressiblytherein a heterologous polynucleotide encoding NR3-1 or NR3-2, and thenrecovering the cultured cells.
 13. A process for obtaining asubstantially homogeneous source of a human NR3 protein according toclaim 12, comprising the subsequent step of obtaining a membranepreparation from the cultured cells.
 14. A method of assaying acandidate ligand for interaction with a human NR3 protein selected fromthe group consisting of the NR3-1 and NR3-2 proteins, which comprisesthe steps of incubating the candidate ligand under appropriateconditions with a cell having incorporated expressibly therein aheterologous polynucleotide encoding NR3-1 or NR3-2, or with a membranepreparation derived therefrom, and then determining the extent ofbinding between the human NR3 protein and the candidate ligand.
 15. Amethod of assaying a candidate ligand for interaction with a humanheteromeric receptor complex comprising an NR3 protein and an NMDAreceptor, which comprises the steps of incubating the candidate ligandunder appropriate conditions with a cell as defined in claim 10, or withmembrane preparation derived therefrom, and then determining the extentof binding between the complex and the candidate ligand, or determiningligand-induced electrical current across said cell or membrane.
 16. Ahuman NR3 protein selected from the group consisting of the NR3-1 andthe NR3-2 proteins, in a form essentially free from other proteins ofhuman origin.
 17. A functional fragment of an NR3 protein selected fromthe group consisting of the NR3-1 and the NR3-2 proteins.
 18. Anantibody which binds a human NR3 protein selected from the groupconsisting of the NR3-1 and the NR3-2 proteins.
 19. An immunogenicfragment of a human NR3 protein selected from the group consisting ofthe NR3-1 and the NR3-2 proteins.
 20. An oligonucleotide comprising atleast about 17 nucleic acids which hybridizes with a polynucleotideencoding an NR3 protein selected from the group consisting of the NR3-1and NRe-2 proteins.